CN111989401A - 猪胰蛋白酶变体 - Google Patents

猪胰蛋白酶变体 Download PDF

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CN111989401A
CN111989401A CN201980026139.4A CN201980026139A CN111989401A CN 111989401 A CN111989401 A CN 111989401A CN 201980026139 A CN201980026139 A CN 201980026139A CN 111989401 A CN111989401 A CN 111989401A
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T·施蒂尔格
S·里瑟姆
C·费勒
A·沃格尔
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Abstract

本发明涉及猪胰蛋白酶的多肽变体、编码这些变体的核酸分子以及包含此类核酸分子的宿主细胞。本发明还涉及这些变体在用于产生胰岛素的方法中的用途。本发明还涉及这些变体作为药剂、作为食品成分或作为饲料成分的用途以及这些变体在制造食品成分或饲料成分的工艺内的用途。

Description

猪胰蛋白酶变体
技术领域
本发明涉及猪胰蛋白酶的多肽变体、编码这些变体的核酸分子以及包含此类核酸分子的宿主细胞。本发明还涉及这些变体在用于产生胰岛素的方法中的用途。本发明还涉及这些变体作为药剂、作为食品成分或作为饲料成分的用途以及这些变体在制造食品成分或饲料成分的工艺内的用途。
背景技术
内肽酶胰蛋白酶用于制造人胰岛素、胰岛素类似物，并且还用于胰岛素衍生物。使用胰蛋白酶通过酶水解由前胰岛素原(PPI)产生人胰岛素、胰岛素类似物或胰岛素衍生物，其中切掉前序列和C肽以便产生相应的产物(参见图1)。胰蛋白酶特异性地在多肽链内的Arg和Lys残基的C末端侧处切割。在人前胰岛素原和甘精前胰岛素原多肽内，在B链和C肽的接点处存在若干Arg和Lys残基，包括B29Lys、B31Arg和B32Arg。因此，在Arg和Lys残基的C末端侧处(包括在B29Lys和B31Arg和B32Arg的C末端侧处)通过胰蛋白酶切割人前胰岛素原和甘精前胰岛素原。对于甘精胰岛素的制造，B32Arg之后进行的胰蛋白酶切割提供最终的甘精胰岛素。B29Lys和B31Arg之后进行的胰岛素酶切割产生副产物desB30-Thr(“des-Thr”)-甘精胰岛素和desB32Arg-甘精胰岛素(也被称为“des-Arg”)。因此，需要去除甘精胰岛素的制造工艺中的副产物。
对于人胰岛素的制造，B31Arg之后进行的胰蛋白酶切割产生B31Arg-人胰岛素(也被称为“mono-Arg”)，并且B32Arg之后进行的胰蛋白酶切割产生B31Arg-B32Arg-人胰岛素(也被称为“di-Arg”)。两者在所述制造工艺中均是中间体。在后续制造工艺中，这两种中间体均转化为最终的人胰岛素。B29Lys之后进行的胰岛素酶切割产生副产物desB30-Thr(“des-Thr”)-人胰岛素。因此，需要去除人胰岛素的制造工艺中的副产物。
对于人胰岛素和甘精胰岛素的制造，B32Arg之后进行的胰蛋白酶切割产生最终的甘精胰岛素，并且对于人胰岛素的制造，其产生中间体，所述中间体在后续工艺步骤中转化为最终的人胰岛素。
为了解决不合需要的desB30-Thr中间体形成的问题，过去的发展产生了猪胰蛋白酶变体S172A，其通过增加针对Arg相对于Lys的C末端侧的选择性来减少此错切割。猪胰蛋白酶变体S172A描述在国际专利申请WO 2007/031187A1中，将其通过引用以其整体特此并入。
现在，在诸位发明人仔细观察胰蛋白酶S172A催化反应的动力学时，他们意外地发现错切割的副产物的形成不仅是由于通过充当内切蛋白酶的胰岛素酶进行的PPI的初级错切割(如文献中所述)，还由于充当外肽酶的猪胰蛋白酶以及其变体S172A所固有的副活性。外肽酶活性还导致在形成des-Arg和des-Thr时甘精胰岛素的降解。
本发明背后的技术问题
因此，现有技术需要具有降低的副活性，尤其是具有降低的外肽酶活性的新型胰蛋白酶变体。此类新型胰蛋白酶变体将减少PPI切割中的副产物的量，并且在由PPI产生胰岛素(或胰岛素衍生物)时将产生更高产率的胰岛素和胰岛素衍生物。
诸位发明人制备了新型猪胰蛋白酶变体，其具有降低的外肽酶活性和/或在PPI切割反应中显示减少的副产物形成。
以上综述不一定描述了本发明解决的所有问题。
发明内容
在第一方面，本发明涉及一种猪胰蛋白酶变体，其包含与SEQ ID NO:1具有至少90％序列同一性的氨基酸序列或者由所述氨基酸序列组成，其中所述氨基酸序列与SEQ IDNO:1相比不同之处至少在于在对应于根据SEQ ID NO:1的天然猪胰蛋白酶的F24、S44、D56、G78、Y131、S172和W193的一个或多个位置处的一个或多个氨基酸取代，
前提是所述氨基酸序列不是根据SEQ ID NO:1的天然猪胰蛋白酶；并且
前提是所述氨基酸序列不是根据SEQ ID NO:2的猪变体胰蛋白酶S172A。
在第二方面，本发明涉及一种核酸分子，其编码根据第一方面的猪胰蛋白酶变体。
在第三方面，本发明涉及一种宿主细胞，其含有根据第二方面的核酸分子。
在第四方面，本发明涉及一种产生根据第一方面的猪胰蛋白酶变体的方法，其包括以下步骤：
培养根据第三方面的宿主细胞，以及从培养基或从所述宿主细胞分离所述猪胰蛋白酶变体。
在第五方面，本发明涉及根据第一方面的猪胰蛋白酶变体或根据第二方面的核酸分子或根据第三方面的宿主细胞，其用作药剂；用作食品成分；用作饲料成分或用于在制造食品成分或饲料成分的工艺内使用。
在第六方面，本发明涉及根据第一方面的猪胰蛋白酶变体在用于产生人胰岛素、胰岛素类似物或胰岛素衍生物的方法中的用途。
在第七方面，本发明涉及根据第一方面的猪胰蛋白酶变体用于切割具有通式A-Lys-Thr-Arg-Arg-B的蛋白质或肽的用途，
其中A是由一个或多个氨基酸组成的氨基酸序列；并且
其中B是由一个或多个氨基酸组成的氨基酸序列。
在第八方面，本发明涉及一种组合物，其包含根据第一方面的猪胰蛋白酶变体或根据第二方面的核酸分子或根据第三方面的宿主细胞。
附图说明
图1示出了人胰岛素和甘精胰岛素的前胰岛素原的主要胰蛋白酶切割位点的示意图。实心三角形代表产生一种或多种产物的切割位点，空心三角形代表产生副产物的切割位点。未显示前胰岛素原的二硫键。
图2示出了用胰蛋白酶变体S172A(SEQ ID NO:2)进行的甘精前胰岛素原的切割。所述图示出了甘精胰岛素的形成以及最主要的切割副产物B31Arg-甘精胰岛素(甘精胰岛素B链的位置31之后的切割，在图中缩写为“des-Arg”)和desB30Thr甘精胰岛素(甘精胰岛素B链的位置29之后的切割，在图中缩写为“des-Thr”)的形成的时间依赖性过程。
图3示出了用胰蛋白酶变体S172A(SEQ ID NO:2)进行的甘精胰岛素的切割，作为胰蛋白酶变体S172A的外肽酶活性的例子。所述图示出了甘精胰岛素的时间依赖性降低以及主要产物B32-desArg-甘精胰岛素(甘精胰岛素B链的位置31之后的切割，在图中缩写为“des-Arg”)和少量副产物B30-desThr甘精胰岛素(甘精胰岛素B链的位置29之后的切割，在图中缩写为“des-Thr”)的形成。
图4示出了用胰蛋白酶变体105号(SEQ ID NO:105)进行的甘精前胰岛素原的切割，作为用经优化的胰蛋白酶变体进行的切割的例子。所述图示出了甘精胰岛素的形成以及最主要的切割副产物B31Arg-甘精胰岛素(甘精胰岛素B链的位置31之后的切割，在图中缩写为“des-Arg”)和desB30Thr甘精胰岛素(甘精胰岛素B链的位置29之后的切割，在图中缩写为“des-Thr”)的形成减少的时间依赖性过程。
具体实施方式
定义
虽然下文详细描述了本发明，但是应理解，本发明不限于所描述的具体方法、方案和试剂，因为这些方法、方案和试剂可以变化。还应理解，本文所用的术语仅出于描述特定实施方案的目的，并不旨在限制本发明的范围，本发明的范围将仅由随附权利要求限定。除非另外定义，否则本文所用的所有技术和科学术语都具有与本发明所属领域的普通技术人员通常所理解相同的含义。
除非另外指示，否则本发明的实践将采用本领域的文献中所解释的化学、生物化学、细胞生物学、免疫学和重组DNA技术的常规方法。
贯穿本说明书和之后的权利要求书，除非上下文另外要求，否则词语“包含(comprise)”和如“包含(comprises)”和“包含(comprising)”等变化形式将理解为暗示包括所述成员、整数或步骤或者成员、整数或步骤的组，但是不排除任何其他成员、整数或步骤或者成员、整数或步骤的组，但是在一些实施方案中，可以排除这样的其他成员、整数或步骤或者成员、整数或步骤的组，即主题在于包括所述成员、整数或步骤或者成员、整数或步骤的组。除非本文另外指示或与上下文明显矛盾，否则在描述本发明的上下文中(尤其在权利要求书的上下文中)所用的术语“一个/一种(a和an)”和“所述”和类似参考应视为同时覆盖单数和复数。在本文中列举值的范围仅旨在用作个别提到落在所述范围内的每个单独值的速记法。除非本文另外指示，否则将每个单独的值并入说明书中，如同在本文中单独列举所述值一样。除非本文另外指示或者另外与上下文明确矛盾，否则本文所述的所有方法都能以任何合适的顺序来进行。在本文中提供的任何和所有例子或示例性语言(例如，“如”)的使用都仅旨在更好地说明本发明，并且不对原本要求保护的本发明的范围施加任何限制。本说明书中的所有语言都不应当视为指示任何未要求保护的要素为本发明实践所必需。
在本说明书的全文中引用了若干文献(例如：专利、专利申请、科学出版物、制造商的说明书、说明书、GenBank登录号序列提交等)。本文的任何内容都不应视为承认本发明因在先发明而无权早于此类披露内容。本文引用的一些文献被表征为“通过引用并入”或“通过引用以其整体并入”。在此类并入的参考文献的定义或教导与本说明书中引用的定义或教导之间发生冲突的情况下，以本说明书的文本为准。
序列：在本文中提及的所有序列都在随附序列表中公开，所述随附序列表及其全部内容和公开内容是本说明书的一部分。
在结合生物材料(如核酸分子、(多)肽、宿主细胞等)使用时，术语“天然存在的”是指在自然界中发现并且未经人工操纵的材料。
如本文所用，术语“氨基酸”或“氨基酸残基”是指天然存在的氨基酸、以与天然存在的氨基酸类似的方式起作用的非天然氨基酸、氨基酸类似物和氨基酸模拟物，全部呈其D和L立体异构体形式，如果其结构允许此类立体异构形式的话。氨基酸在本文中以其名称、其通常已知的三字母符号或IUPAC-IUB生物化学命名委员会推荐的单字母符号提及。
在结合氨基酸使用时，术语“天然存在的”是指20种常规氨基酸(即，丙氨酸(A)、半胱氨酸(C)、天冬氨酸(D)、谷氨酸(E)、苯丙氨酸(F)、甘氨酸(G)、组氨酸(H)、异亮氨酸(I)、赖氨酸(K)、亮氨酸(L)、甲硫氨酸(M)、天冬酰胺(N)、脯氨酸(P)、谷氨酰胺(Q)、精氨酸(R)、丝氨酸(S)、苏氨酸(T)、缬氨酸(V)、色氨酸(W)和酪氨酸(Y))以及硒代半胱氨酸、吡咯赖氨酸(PYL)和吡咯啉-羧基赖氨酸(PCL)。术语“天然存在的氨基酸”和“可编码氨基酸”在本文中可互换使用。
如本文所用，术语“非天然氨基酸”意在指代不是天然编码的或在任何生物体的遗传密码中发现的氨基酸。
如本文所用，术语“氨基酸类似物”是指与天然存在的氨基酸具有相同的基础化学结构的化合物。
如本文所用，术语“氨基酸模拟物”是指具有与氨基酸的通用化学结构不同的结构但是以与天然存在的氨基酸类似的方式起作用的化学化合物。
如本文所用，术语“肽”是指任何长度的氨基酸的多聚体形式，其例如包含2个或更多个、或者3个或更多个、或者4个或更多个、或者6个或更多个、或者8个或更多个、或者9个或更多个、或者10个或更多个、或者13个或更多个、或者16个或更多个、或者21个或更多个通过肽键共价连接的氨基酸。术语“多肽”是指大型肽，特别地是指具有超过100个氨基酸残基的肽。术语“多肽”和“蛋白质”在本文中可互换使用。本申请提供了本文公开的若干肽和多肽的氨基酸序列信息。在本文中从肽或多肽的氨基末端(在左侧)向羧基末端(在右侧)呈现氨基酸序列。
如本文所用，(多)肽的“变体”是指与对应的参考(多)肽相比不同之处在于其氨基酸序列中的一个或多个氨基酸修饰的(多)肽。在一个实施方案中，“变体”维持参考(多)肽的一种或多种生物活性。参考(多)肽可以是野生型(多)肽或非天然存在的(多)肽。术语“变体”和“突变体”在本文中可互换使用。
如本文所用，术语“猪胰蛋白酶变体”是指与如SEQ ID NO:1中所示的野生型猪胰蛋白酶相比不同之处在于其氨基酸序列中的一个或多个氨基酸修饰的多肽。术语“猪胰蛋白酶变体”具体地是指与如SEQ ID NO:1中所示的野生型猪胰蛋白酶相比不同之处在于一个或多个氨基酸交换的多肽。根据本发明，“猪胰蛋白酶变体”表现出与参考胰蛋白酶(例如，根据SEQ ID NO:1的野生型猪胰蛋白酶或根据SEQ ID NO:2的胰蛋白酶变体S172A)的内切蛋白水解活性类似的内切蛋白水解活性，但是“猪胰蛋白酶变体”和参考胰蛋白酶的内切蛋白水解活性在其针对某些切割位点的选择性方面可以不同。
术语“氨基酸修饰”涵盖氨基酸交换、氨基酸缺失和氨基酸添加。术语“氨基酸交换”和“氨基酸取代”在本文中可互换使用。术语“氨基酸缺失”涵盖N末端截短、内部缺失和C末端截短。术语“氨基酸添加”涵盖N末端添加、氨基酸插入和C末端添加。
表述“Xaa24”(和类似表述，如Xaa44、Xaa56、Xaa78等)是指给定氨基酸序列的位置24(或相应地44、56、78等)处的可变氨基酸。与本申请一起提供的序列表含有具有可变氨基酸位置的一个蛋白质序列，即SEQ ID NO:3。根据SEQ ID NO:3的蛋白质序列内的可变氨基酸位置由氨基酸Xaa指示(呈三字母代码形式)。此例子中的数字24指示此可变位置是根据SEQ ID NO:3的蛋白质序列的第24个氨基酸。同样，表述Xaa44指示SEQ ID NO:3的第44个位置是可变氨基酸位置，以此类推。
如本文所用，术语“人胰岛素”是指其结构和特性是熟知的人激素。人胰岛素具有两条通过半胱氨酸残基之间的二硫桥连接的肽链(A链和B链)，即A链和B链。A链是21个氨基酸的肽并且B链是30个氨基酸的肽，两条链通过三个二硫桥连接：一个在A链的位置6和11中的半胱氨酸之间；第二个在A链的位置7中的半胱氨酸与B链的位置7中的半胱氨酸之间；并且第三个在A链的位置20中的半胱氨酸与B链的位置19中的半胱氨酸之间。
如本文所用，术语“胰岛素的衍生物”和“胰岛素衍生物”是指具有如下分子结构的肽，所述分子结构在形式上可以衍生自天然存在的胰岛素的结构(例如人胰岛素的结构)，其中一个或多个有机取代基(例如，脂肪酸)与一个或多个氨基酸结合。任选地，天然存在的胰岛素中存在的一个或多个氨基酸可能已被缺失和/或被其他氨基酸(包括不可编码的氨基酸)替代，或者氨基酸(包括不可编码的氨基酸)已被添加到天然存在的胰岛素中。胰岛素衍生物的例子包括但不限于以下：
(i).“地特胰岛素”，其与人胰岛素相比不同之处在于位置B30中的C末端苏氨酸被去除，并且脂肪酸残基(肉豆蔻酸残基)连接到位置B29中的赖氨酸的ε氨基官能团。
(ii).“德谷胰岛素”，其与人胰岛素相比不同之处在于最后一个氨基酸从B链缺失并且向十六烷二酸添加来自B29Lys的谷氨酰基连接。
如本文所用，术语“胰岛素的类似物”和“胰岛素类似物”是指具有如下分子结构的肽，所述分子结构可以通过缺失和/或交换在天然存在的胰岛素中存在的至少一个氨基酸残基和/或添加至少一个氨基酸残基而在形式上衍生自天然存在的胰岛素的结构(例如人胰岛素的结构)。添加的和/或交换的氨基酸残基可以是可编码的氨基酸残基或另一个天然存在的残基或纯合成的氨基酸残基。胰岛素类似物的例子包括但不限于以下：
(i).“门冬胰岛素”通过重组DNA技术产生，使得人胰岛素中的作为脯氨酸的氨基酸B28(即，人胰岛素的B链中的氨基酸28号)被天冬氨酸替代；
(ii).“赖脯胰岛素”通过重组DNA技术产生，使得人胰岛素的B链的C末端上的倒数第二个赖氨酸和脯氨酸残基互换(人胰岛素：B28Pro-B29Lys；赖脯胰岛素：B28Lys-B29Pro)；
(iii).“赖谷胰岛素”与人胰岛素相比不同之处在于位置B3处的氨基酸天冬酰胺被赖氨酸替代，并且位置B29中的赖氨酸被谷氨酸替代；
(iv).“甘精胰岛素”与人胰岛素相比不同之处在于位置A21处的天冬酰胺被甘氨酸替代，并且B链在羧基末端延伸了两个精氨酸。
如本文所用，术语“产率”是指胰蛋白酶切割之后获得的所得产物(即，胰岛素或胰岛素衍生物或胰岛素类似物)与胰蛋白酶切割之前的前胰岛素原(或前胰岛素原衍生物或前胰岛素原类似物)的比率。产率依赖于胰蛋白酶变体在所需切割位点处进行切割的选择性及其核酸外切酶活性。胰蛋白酶变体仅在所需切割位点处进行切割的选择性越高，并且其核酸外切酶活性越低，产率越高。
例如，“甘精胰岛素的产率”或“B31Arg-B32Arg-人胰岛素的产率”是指胰蛋白酶切割之后获得的所得甘精胰岛素或B31Arg-B32Arg-人胰岛素分别与胰蛋白酶切割之前的甘精前胰岛素原或人前胰岛素原的比率。“甘精胰岛素的产率”和“B31Arg-B32Arg-人胰岛素的产率”依赖于胰蛋白酶变体仅在B32Arg之后进行切割的选择性及其核酸外切酶活性。胰蛋白酶变体仅在B32Arg之后进行切割的选择性越高，并且其核酸外切酶活性越低，产率越高。
如本文所用，术语“增加的选择性”是指与参考胰蛋白酶相比，胰蛋白酶变体在不合需要的切割位点处以更小程度进行切割并且在所需切割位点处以更高程度进行切割的特性。增加的选择性导致不合需要的副产物的形成减少。参考胰蛋白酶可以是例如根据SEQID NO:1的野生型猪胰蛋白酶或根据SEQ ID NO:2的胰蛋白酶变体S172A。
两个氨基酸序列之间的“序列同一性”指示序列之间相同的氨基酸的百分比。除人工产生以外，可以通过以下手段产生用于比较的序列的最佳比对：Smith和Waterman,1981,Adv.App.Math.2,482-489的局部同源性算法(通过引用随同并入)，Needleman和Wunsch,1970,J.Mol.Biol.48,443-453的局部同源性算法(通过引用随同并入)，Pearson和Lipman,1988,Proc.Natl Acad.Sci.USA 85,2444-2448的相似性搜索方法(通过引用随同并入)，或使用这些算法的计算机程序(Wisconsin Genetics Software Package,GeneticsComputer Group,575Science Drive,Madison,Wis.中的GAP、BESTFIT、FASTA、BLAST P、BLAST N和TFASTA)。
在本发明的上下文中，如果没有另外明确说明，则通常相对于未修饰序列的总长度来计算修饰序列与其衍生自的序列之间的序列同一性程度。在实施方案中，在任何序列都不是修饰序列的情况下，除非另外说明，否则相对于定义为参考序列的序列的总长度来计算序列同一性程度。在实施方案中，在任何序列都不能被视为参考序列的情况下，除非另外说明，否则相对于两个序列中的较长者来计算序列同一性程度。
根据本发明的一些实施方案的“核酸分子”是脱氧核糖核酸(DNA)或核糖核酸(RNA)。根据本发明的核酸分子可以呈是单链或双链的分子形式；并且可以为线性的或共价闭合以形成环。
术语“DNA”是指包含脱氧核糖核苷酸残基并且可以完全或基本上由脱氧核糖核苷酸残基构成的分子。“脱氧核糖核苷酸”是指在β-D-呋喃核糖基的2’-位置缺少羟基的核苷酸。术语“DNA”包含经分离的DNA，如部分或完全纯化的DNA、基本上纯的DNA、合成DNA和重组产生的DNA，并且包括经修饰的DNA，其与天然存在的DNA相比不同之处在于一个或多个核苷酸的添加、缺失、取代和/或改变。此类改变可以包括添加非核苷酸材料，如添加到DNA的一个或多个末端或添加到内部，例如在DNA的一个或多个核苷酸处添加。DNA分子中的核苷酸还可以包含非标准核苷酸，如非天然存在的核苷酸或化学合成的核苷酸。这些经改变的DNA可以被称为类似物或天然存在的DNA的类似物。在结合核苷酸使用时，术语“天然存在的”是指碱基腺嘌呤(A)、胞嘧啶(C)、鸟嘌呤(G)、胸腺嘧啶(T)和尿嘧啶(U)。
术语“RNA”是指包含核糖核苷酸残基并且可以完全或基本上由核糖核苷酸残基构成的分子。“核糖核苷酸”是指在β-D-呋喃核糖基的2’-位置具有羟基的核苷酸。术语“RNA”包含经分离的RNA，如部分或完全纯化的RNA、基本上纯的RNA、合成RNA和重组产生的RNA，并且包括经修饰的RNA，其与天然存在的RNA相比不同之处在于一个或多个核苷酸的添加、缺失、取代和/或改变。此类改变可以包括添加非核苷酸材料，如添加到RNA的一个或多个末端或添加到内部，例如在RNA的一个或多个核苷酸处添加。RNA分子中的核苷酸还可以包含非标准核苷酸，如非天然存在的核苷酸或化学合成的核苷酸或脱氧核苷酸。这些经改变的RNA可以被称为类似物或天然存在的RNA的类似物。根据本发明，“RNA”是指单链RNA或双链RNA。在一个实施方案中，RNA是mRNA，例如体外经转录RNA(IVT RNA)或合成RNA。RNA还可以被修饰，例如具有一个或多个提高RNA稳定性(例如，半衰期)的修饰。此类修饰是本领域技术人员已知的，并且包括例如5’-帽或5’帽类似物。
根据本发明的核酸分子可以含在/包含在载体中。如本文所用，术语“载体”包括技术人员已知的所有载体，包括质粒载体、粘粒载体、噬菌体载体(如λ噬菌体)、病毒载体(如腺病毒或杆状病毒载体)或人工染色体载体(如细菌人工染色体(BAC)、酵母人工染色体(YAC)或P1人工染色体(PAC))。所述载体包括表达以及克隆载体。表达载体包含质粒以及病毒载体，并且通常含有所需编码序列和在特定宿主生物体(例如，细菌、酵母、植物、昆虫或哺乳动物)中或在体外表达系统中表达可操作地连接的编码序列所需的适当DNA序列。克隆载体通常用于工程化和扩增某一所需DNA片段，并且可能缺少表达所需DNA片段所需的功能性序列。
可替代地，可以将根据本发明的核酸分子整合到基因组(例如，宿主细胞的基因组)中。将特定核酸分子整合到基因组中的手段和方法是本领域技术人员已知的。
术语“细胞”或“宿主细胞”通常是指完整细胞，即具有完整膜并且尚未释放其正常细胞内组分(如酶、细胞器或遗传材料)的细胞。完整细胞通常是活细胞，即能够执行其正常代谢功能的活细胞。如本文所用，所述术语涉及可以用外源核酸转染或转化的任何细胞。在一些实施方案中，在经外源核酸转染或转化并转移至接受者时，细胞可以在接受者体内表达所述核酸。术语“细胞”包括原核细胞，如细菌细胞；以及真核细胞，如酵母细胞、真菌细胞或哺乳动物细胞。细菌细胞包括来自以下的细胞：革兰氏阴性细菌菌株，如大肠杆菌(Escherichia coli)、变形杆菌属(Proteus)和假单胞菌属(Pseudomonas)的菌株；以及革兰氏阳性细菌菌株，如芽孢杆菌属(Bacillus)、链霉菌属(Streptomyces)、葡萄球菌属(Staphylococcus)和乳球菌属(Lactococcus)的菌株。真菌细胞包括来自以下物种的细胞：木霉属(Trichoderma)、链孢霉属(Neurospora)和曲霉菌属(Aspergillus)。酵母细胞包括来自以下物种的细胞：酵母属(Saccharomyces)(例如，酿酒酵母(Saccharomycescerevisiae))、裂殖酵母属(Schizosaccharomyces)(例如，粟酒裂殖酵母(Schizosaccharomyces pombe))、毕赤酵母属(Pichia)(例如，巴斯德毕赤酵母(Pichiapastoris)和甲醇毕赤酵母(Pichia methanolica))、驹形氏酵母(Komagataella)(例如，巴斯德驹形氏酵母(Komagataella pastoris)和法夫驹形氏酵母(Komagataella phaffii))和汉逊酵母属(Hansenula)。哺乳动物细胞包括例如CHO细胞、BHK细胞、HeLa细胞、COS细胞、HEK293等。也可以使用两栖动物细胞、昆虫细胞、植物细胞和本领域中用于表达异源蛋白质的任何其他细胞。哺乳动物细胞特别适用于过继转移，如来自人、小鼠、仓鼠、猪、山羊和灵长类动物的细胞。所述细胞可以衍生自众多种组织类型，并且包括原代细胞和细胞系，如免疫系统的细胞，特别是抗原呈递细胞，如树突细胞和T细胞；干细胞，如造血干细胞和间充质干细胞；和其他细胞类型。抗原呈递细胞是在其表面上展示在主要组织相容性复合物的背景下的抗原的细胞。T细胞可以使用其T细胞受体(TCR)识别此复合物。“细胞”或“宿主细胞”可以是经分离的或者组织或生物体(特别是“非人生物体”)的部分。
如本文所用，术语“非人生物体”意在包括非人灵长类动物或其他动物，特别是哺乳动物，如牛、马、猪、绵羊、山羊、狗、猫、兔或啮齿类动物(如小鼠、大鼠、豚鼠和仓鼠)。
如本文所用，术语“药剂”是指用于疗法(即，用于治疗疾病或障碍)的物质/组合物。
根据本发明，术语“疾病或障碍”是指任何病理或不健康状态。
“治疗”意指向受试者给予化合物或组合物或者化合物或组合物的组合，以便预防或消除疾病或障碍；阻止或减慢受试者的疾病或障碍；抑制或减慢新生疾病或障碍在受试者体内的发展；在目前患有或先前曾患有疾病或障碍的受试者中降低症状和/或复发的频率或严重程度；和/或延长(即，增加)受试者的寿命。
具体地，术语“治疗(treating/treatment)疾病或障碍”包括治愈疾病或障碍或其症状，缩短疾病或障碍或其症状的持续时间，改善、预防、减慢或抑制疾病或障碍或其症状的进展或恶化，或预防或延迟疾病或障碍或其症状的发作。
根据本发明，术语“受试者”意指用于治疗的受试者，特别是患病受试者(也被称为“患者”)，包括人、非人灵长类动物或其他动物，特别是哺乳动物，如牛、马、猪、绵羊、山羊、狗、猫、兔或啮齿类动物(如小鼠、大鼠、豚鼠和仓鼠)。在一个实施方案中，受试者/患者是人。
根据本发明的“组合物”含有有效量的本发明的猪胰蛋白酶变体(在本文中也被称为“活性剂”)。根据本发明的组合物可以(a)用于制造工艺中，特别是用于制备肽的制造工艺中，特别是用于制备人胰岛素、胰岛素衍生物和/或胰岛素类似物的制造工艺中；(b)用作食品成分；(c)用作饲料成分；(d)或用于在制造食品成分或饲料成分的工艺内使用。根据本发明的组合物也可以用于治疗疾病或障碍，并且因此可以是药物组合物。
根据本发明的“药物组合物”含有治疗有效量的本发明的猪胰蛋白酶变体、核酸分子或宿主细胞(在本文中也被称为“活性剂”)以产生所需反应或所需效果。根据本发明的药物组合物还可以包含至少一种其他活性剂。
在一个实施方案中，组合物以均匀剂型提供，并且能以本身已知的方式制备。组合物可以例如呈溶液或悬浮液的形式。
组合物或药物组合物还可以包含一种或多种赋形剂。在药物组合物的一个实施方案中，所述一种或多种赋形剂全部是药学上可接受的。如本文所用，“药学上可接受的”是指哺乳动物或人在生理上良好耐受的。具体地，它意指由联邦政府或州政府的监管机构批准的或在美国药典或其他普遍认可的药典中列出的用于在动物体内、更特别地在人体内使用。
根据本发明的组合物或药物组合物能以组合物的形式存在，其中不同的活性剂和赋形剂(例如，稀释剂和/或载体)彼此混合；或者可以采取组合制剂的形式，其中活性剂以部分或完全不同的形式存在。这种组合或组合制剂的例子是部件试剂盒(kit-of-parts)。
如果上下文中没有另外说明，则术语“活性剂”和“活性成分”是指本发明的猪胰蛋白酶变体、本发明的核酸分子和本发明的宿主细胞。术语“活性剂”和“活性成分”在本文中可互换使用。
术语“载体”是指天然或合成性质的有机或无机组分，其中组合活性剂以便促进、增强或实现应用。根据本发明，术语“载体”还包括一种或多种相容的固体或液体填充剂、稀释剂、佐剂、赋形剂、媒介物或囊封物质。此类载体可以是无菌液体，如在水中的盐水溶液以及油，所述油包括石油、动物、植物或合成来源的那些，如花生油、大豆油、矿物油、芝麻油等。盐水溶液和水性右旋糖和甘油溶液也可以用作液体载体。合适的赋形剂包括淀粉、葡萄糖、乳糖、蔗糖、明胶、麦芽、稻、面粉、白垩、硅胶、硬脂酸钠、甘油单硬脂酸酯、滑石、氯化钠、脱脂乳粉、甘油、丙烯、乙二醇、水、乙醇等。如果需要，组合物或药物组合物还可以含有少量的湿润剂或乳化剂或pH缓冲剂。这些组合物可以采取溶液、悬浮液、乳液、片剂、丸剂、胶囊、粉末、缓释配制品等形式。组合物或药物组合物可以用传统的粘合剂和载体(如甘油三酯)配制为栓剂。本发明的化合物可以配制为中性或盐形式。药学上可接受的盐包括与游离氨基形成的那些盐，如衍生自盐酸、磷酸、乙酸、草酸、酒石酸等的那些；以及与游离羧基形成的那些盐，如衍生自钠、钾、铵、钙、氢氧化铁、异丙胺、三乙胺、2-乙基氨基乙醇、组氨酸、普鲁卡因等的那些。此类组合物将含有有效量的化合物(在一些实施方案中呈纯化形式)以及合适量的载体。配制品应当符合给予模式。
如本文所用，术语“赋形剂”旨在包括可以存在于组合物或药物组合物中并且不是活性剂的所有物质，如盐、粘合剂(例如，乳糖、右旋糖、蔗糖、海藻糖、山梨醇、甘露醇)、载体、润滑剂、增稠剂、表面活性剂、防腐剂(例如，抗氧化剂、柠檬酸、柠檬酸钠、苯扎氯铵、氯丁醇、半胱氨酸、甲硫氨酸、对羟基苯甲酸酯、硫柳汞)、乳化剂、缓冲物质、调味剂或着色剂。
盐被包括在本发明中，并且也可以是药学上可接受的盐。这种盐或药学上可接受的盐以非限制性方式包含从以下酸制备的那些盐：盐酸、氢溴酸、硫酸、硝酸、磷酸、马来酸、乙酸、水杨酸、柠檬酸、甲酸、丙二酸、琥珀酸等。盐和药学上可接受的盐也可以制备为碱金属盐或碱土金属盐，如钠盐、钾盐或钙盐。可以添加盐以调节离子强度或张力。
用于在根据本发明的组合物或药物组合物中使用的合适的缓冲物质包括呈盐形式的乙酸、呈盐形式的柠檬酸、呈盐形式的硼酸和呈盐形式的磷酸。
组合物或药物组合物也可以配制为用适当稀释剂重构的稳定冻干产物，其任选地包含如上文所定义的一种或多种赋形剂。
本文所述的活性剂和药物组合物能以治疗有效量来给予。“治疗有效量”是指单独或与其他剂量一起实现所需治疗反应或所需治疗效果、在一个实施方案中而不引起不可接受的副作用的量。在治疗特定疾病或特定病症的情形中，所需反应可以与抑制病程相关。这包括减慢病程，特别是中断或逆转病程。疾病或病症的治疗中的所需反应也可以是延迟所述疾病或所述病症的发作或者预防所述疾病或所述病症的发作。本文所述的药剂或组合物的有效量将取决于待治疗的病症、疾病的严重程度、受试者的个别参数(包括年龄、生理状况、体型和体重)、治疗的持续时间、伴随治疗(如果存在)的类型、具体给予途径和类似因素。因此，所给予的本文所述的药剂的剂量可以取决于若干此类参数。在初始剂量引起的受试者反应不足的情形中，可以使用较高剂量(或通过不同的更集中的给予途径实现的有效较高的剂量)。
如本文所用，术语“部件试剂盒(简写：试剂盒)”是指包含一个或多个容器以及任选地数据载体的制品。所述一个或多个容器可以填充有一种或多种上文所提到的药剂(试剂)。另外的容器可以被包括在试剂盒中，所述另外的容器含有例如稀释剂、缓冲液和其他试剂。所述数据载体可以是非电子数据载体，例如图形数据载体，如信息传单、信息表、条码或存取码；或电子数据载体，如光盘(CD)、数字多功能光盘(DVD)、微芯片或另一种基于半导体的电子数据载体。存取码可以容许访问数据库，例如互联网数据库、集中式数据库或分布式数据库。所述数据载体可以包含使用本发明的活性剂的说明书。例如，数据载体可以包含(a)在制造工艺中，特别是在制备肽的制造工艺中，特别是在制备人胰岛素、胰岛素衍生物和/或胰岛素类似物的制造工艺中；(b)作为食品成分；(c)作为饲料成分；(d)或在制造食品成分或饲料成分的工艺内使用活性剂的说明书。
如本文所用，术语“前胰岛素原”或缩写“PPI”是指单链胰岛素前体，其以前序列从N末端部分开始，随后是B链、C肽和A链(参见图1)。
本发明的实施方案
在以下段落中，将描述本发明的某些要素。这些要素可以用具体实施方案来列举，然而应当理解，其能以任何方式和任何数目组合以产生另外的实施方案。差异性描述的实施例和实施方案不应当视为将本发明限制于仅明确描述的实施方案。本说明书应当理解为支持并涵盖组合明确描述的实施方案与任何数目的所公开要素的实施方案。此外，除非上下文另外指示，否则应当考虑本申请的说明书公开本申请中所有所述要素的任何排列和组合。
在第一方面，本发明涉及猪胰蛋白酶变体，其包含与SEQ ID NO:1具有至少90％序列同一性的氨基酸序列或者由所述氨基酸序列组成，其中所述氨基酸序列与SEQ ID NO:1相比不同之处至少在于在对应于根据SEQ ID NO:1的天然猪胰蛋白酶的F24、S44、D56、G78、Y131、S172和W193的一个或多个位置处的一个或多个氨基酸取代，
前提是所述氨基酸序列不是根据SEQ ID NO:1的天然猪胰蛋白酶；并且
前提是所述氨基酸序列不是根据SEQ ID NO:2的猪变体胰蛋白酶S172A。
在第一方面的一个实施方案中，
对应于F24的位置处的氨基酸未被Asp、Glu或Gly取代；
对应于S44的位置处的氨基酸未被Tyr取代；
对应于Y131的位置处的氨基酸未被Lys或Pro取代；并且/或者
对应于W193的位置处的氨基酸未被Asn或Cys取代。
在第一方面的一个实施方案中，
对应于F24的位置处的氨基酸被选自以下的氨基酸取代：Ala、Asn、Arg、Gln、Ile、Leu、Lys、Met、Ser、Thr和Val；
对应于S44的位置处的氨基酸被选自以下的氨基酸取代：Leu和Pro；
对应于D56的位置处的氨基酸被选自以下的氨基酸取代：Ala、Asn、His和Trp；
对应于G78的位置处的氨基酸被选自以下的氨基酸取代：Ala、Glu、Pro、Ser和Tyr；
对应于Y131的位置处的氨基酸被选自以下的氨基酸取代：Ala、Asn、Asp、Cys、Gln、Glu、Gly、His、Ile、Leu、Met、Ser、Thr、Trp和Val；
对应于S172的位置处的氨基酸被选自以下的氨基酸取代：Ala、Cys和Thr；并且/或者
对应于W193的位置处的氨基酸被选自以下的氨基酸取代：Phe、Ser、Thr和Tyr。
在第一方面的进一步的实施方案中，
对应于F24的位置处的氨基酸被Arg取代；
对应于S44的位置处的氨基酸被Pro取代；
对应于D56的位置处的氨基酸被His取代；
对应于G78的位置处的氨基酸被Pro取代；
对应于Y131的位置处的氨基酸被Met取代；
对应于S172的位置处的氨基酸被Ala取代；并且/或者
对应于W193的位置处的氨基酸被Ser取代。
在第一方面的一个实施方案中，所述氨基酸序列与SEQ ID NO:1相比另外的不同之处至少在于在对应于根据SEQ ID NO:1的天然猪胰蛋白酶的R99、R107、K125和K170的一个或多个位置处的一个或多个氨基酸取代。
在第一方面的一个实施方案中，
对应于R107的位置处的氨基酸未被Pro取代；并且/或者
对应于K170的位置处的氨基酸未被Ile或Phe取代。
在第一方面的进一步的实施方案中，
对应于R99的位置处的氨基酸被选自以下的氨基酸取代：Ala、Asn、Asp、Glu、Gly、His、Leu、Phe、Thr、Trp和Tyr；
对应于R107的位置处的氨基酸被选自以下的氨基酸取代：Asp、Gly、Pro、Ser和Thr；
对应于K125的位置处的氨基酸被选自以下的氨基酸取代：Ala、Cys、Gln、Glu、Gly、His、Leu、Ser和Tyr；并且/或者
对应于K170的位置处的氨基酸被选自以下的氨基酸取代：Ala、Asn、Gly和Tyr。
在第一方面的进一步的实施方案中，
对应于R99的位置处的氨基酸被选自以下的氨基酸取代：Ala、His和Asn；
对应于R107的位置处的氨基酸被Thr取代；
对应于K125的位置处的氨基酸被选自以下的氨基酸取代：Ala、Cys和Ser；并且/或者
对应于K170的位置处的氨基酸被Ala取代。
在第一方面的一个实施方案中，所述氨基酸序列与根据SEQ ID NO:1的猪野生型胰蛋白酶具有至少91％或至少92％或至少93％或至少94％或至少95％或至少96％或至少97％或至少98％或至少99％序列同一性。
在第一方面的一个实施方案中，所述氨基酸序列与根据SEQ ID NO:1的猪野生型胰蛋白酶相比不同之处在于1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22或23个氨基酸交换。
在第一方面的一个实施方案中，所述氨基酸序列是SEQ ID NO:3，其中
Xaa24是选自以下的氨基酸：Ala、Asn、Arg、Gln、Ile、Leu、Lys、Met、Phe、Ser、Thr和Val；
Xaa44是选自以下的氨基酸：Leu、Pro和Ser；
Xaa56是选自以下的氨基酸：Ala、Asn、Asp、His和Trp；
Xaa78是选自以下的氨基酸：Ala、Glu、Gly、Pro、Ser和Tyr；
Xaa99是选自以下的氨基酸：Ala、Arg、Asn、Asp、Glu、Gly、His、Leu、Phe、Thr、Trp和Tyr；
Xaa107是选自以下的氨基酸：Arg、Asp、Gly、Pro、Ser和Thr；
Xaa125是选自以下的氨基酸：Ala、Cys、Gln、Glu、Gly、His、Leu、Lys、Ser和Tyr；
Xaa131是选自以下的氨基酸：Ala、Asn、Asp、Cys、Gln、Glu、Gly、His、Ile、Leu、Met、Ser、Thr、Trp、Tyr和Val；
Xaa170是选自以下的氨基酸：Ala、Asn、Gly、Lys和Tyr；
Xaa172是选自以下的氨基酸：Ala、Cys、Ser和Thr；并且/或者
Xaa193是选自以下的氨基酸：Phe、Ser、Thr、Trp和Tyr；
前提是SEQ ID NO:3不是根据SEQ ID NO:1的猪野生型胰蛋白酶；并且
前提是SEQ ID NO:3不是根据SEQ ID NO:2的猪变体胰蛋白酶S172A。
在第一方面的进一步的实施方案中，所述氨基酸序列是SEQ ID NO:3，其中
Xaa24是选自以下的氨基酸：Phe和Arg；
Xaa44是选自以下的氨基酸：Ser和Pro；
Xaa56是选自以下的氨基酸：Asp和His；
Xaa78是选自以下的氨基酸：Gly和Pro；
Xaa99是选自以下的氨基酸：Ala、Arg、His和Asn；
Xaa107是选自以下的氨基酸：Arg和Thr；
Xaa125是选自以下的氨基酸：Ala、Cys、Lys和Ser；
Xaa131是选自以下的氨基酸：Met和Tyr；
Xaa170是选自以下的氨基酸：Ala和Lys；
Xaa172是选自以下的氨基酸：Ala和Ser；并且/或者
Xaa193是选自以下的氨基酸：Ser和Trp；
前提是SEQ ID NO:3不是根据SEQ ID NO:1的猪野生型胰蛋白酶；并且
前提是SEQ ID NO:3不是根据SEQ ID NO:2的猪变体胰蛋白酶S172A。
在第一方面的一个实施方案中，所述氨基酸序列选自SEQ ID NO:4至119。在一个实施方案中，所述氨基酸序列选自SEQ ID NO:89、SEQ ID NO:90、SEQ ID NO:91、SEQ IDNO:92、SEQ ID NO:93、SEQ ID NO:94、SEQ ID NO:95、SEQ ID NO:96、SEQ ID NO:97和SEQID NO:98。
在第一方面的一个实施方案中，所述变体能够切割具有通式A-Lys-Thr-Arg-Arg-B的肽，以产生通式A-Lys-Thr-Arg-Arg的切割产物，产率为至少80％(例如，至少81％、至少82％、至少83％、至少84％、至少85％、至少86％、至少87％、至少88％、至少89％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％)，
其中A是由一个或多个氨基酸组成的氨基酸序列；并且
其中B是由一个或多个氨基酸组成的氨基酸序列。
在第一方面的进一步的实施方案中，A是由1-105个氨基酸(例如，1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、30、35、40、45、50、55、60、65、70、75、80、85、90、95、100或105个氨基酸)组成的氨基酸序列，并且B是由1-105个氨基酸(例如，1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、30、35、40、45、50、55、60、65、70、75、80、85、90、95、100或105个氨基酸)组成的氨基酸序列。
在第一方面的一个实施方案中，与根据SEQ ID NO:2的猪变体胰蛋白酶S172A相比，变体表现出在位置B32-Arg的C末端的位置处对人前胰岛素原、甘精前胰岛素原、赖脯前胰岛素原、门冬前胰岛素原或赖谷前胰岛素原的切割的增加的选择性。
在本发明的第一方面的一些实施方案中，猪胰蛋白酶变体是冻干的。
在第二方面，本发明涉及核酸分子，其编码
-如本发明的第一方面中所定义的猪胰蛋白酶变体；或者
-如本发明的第一方面中所定义的猪胰蛋白酶变体的胰蛋白酶原前体分子。
在第二方面的一个实施方案中，所述核酸分子包含在载体中或整合到基因组中。
在第三方面，本发明涉及宿主细胞，其含有如第二方面中所定义的核酸分子。在一个实施方案中，宿主细胞是重组宿主细胞。
在第四方面，本发明涉及产生本发明的猪胰蛋白酶变体的方法，其包括以下步骤：
培养根据第三方面的宿主细胞；以及
从培养基或从宿主细胞分离猪胰蛋白酶变体的胰蛋白酶原前体分子。
在第四方面的一个实施方案中，所述方法包括进一步的步骤：
活化胰蛋白酶原前体分子，从而获得所述猪胰蛋白酶变体。
胰蛋白酶原前体分子的活化可以化学地或酶促地实现。例如，可以调节pH，使得发生自体活化。在另一个例子中，可以添加蛋白酶(例如，肠激酶)以酶促地活化胰蛋白酶原。
在第五方面，本发明涉及本发明的猪胰蛋白酶变体或本发明的核酸分子或本发明的宿主细胞，其用作药剂；用作食品成分；用作饲料成分或用于在制造食品成分或饲料成分的工艺内使用。
第五方面的一个实施方案涉及本发明的猪胰蛋白酶变体，其用作食品成分；用作饲料成分或用于在制造食品成分或饲料成分的工艺内使用。
在第五方面的一个实施方案中，药剂用于在治疗肿胀(尤其是由创伤引起的肿胀)、炎症(尤其是由创伤引起的炎症)、血栓性静脉炎、活动性关节病、消化酶缺乏(例如，由胰腺功能不全引起)和/或消化紊乱中使用。
换言之，本发明涉及本发明的猪胰蛋白酶变体或本发明的核酸分子或本发明的宿主细胞，其用于在治疗肿胀(尤其是由创伤引起的肿胀)、炎症(尤其是由创伤引起的炎症)、血栓性静脉炎、活动性关节病、消化酶缺乏(例如，由胰腺功能不全引起)和/或消化紊乱中使用。
关于用作食品成分、用作饲料成分或用于制造工艺内，第五方面可以可替代地如下表述：
本发明的猪胰蛋白酶变体作为食品成分的用途。
本发明的猪胰蛋白酶变体作为饲料成分的用途。
本发明的猪胰蛋白酶变体在制造食品成分或饲料成分的工艺中的用途。
在第六方面，本发明涉及本发明的猪胰蛋白酶变体在用于产生人胰岛素、胰岛素类似物或胰岛素衍生物的方法中的用途。
第六方面涵盖本发明的猪胰蛋白酶变体在用于产生人胰岛素、胰岛素类似物或胰岛素衍生物的体外方法和体内方法两者中的用途。
关于在体内方法中的使用，第六方面可以可替代地如下表述：
根据第一方面的猪胰蛋白酶变体，其用于在人胰岛素、胰岛素类似物或胰岛素衍生物的体内产生中使用。
在第六方面的一个实施方案中，胰岛素类似物选自门冬胰岛素、赖脯胰岛素、赖谷胰岛素和甘精胰岛素。
在第七方面，本发明涉及本发明的猪胰蛋白酶变体用于切割具有通式A-Lys-Thr-Arg-Arg-B的蛋白质或肽的用途，
其中A是由一个或多个氨基酸组成的氨基酸序列；并且
其中B是由一个或多个氨基酸组成的氨基酸序列。
在第七方面的进一步的实施方案中，A是由1-105个氨基酸(例如，1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、30、35、40、45、50、55、60、65、70、75、80、85、90、95、100或105个氨基酸)组成的氨基酸序列，并且B是由1-105个氨基酸(例如，1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、30、35、40、45、50、55、60、65、70、75、80、85、90、95、100或105个氨基酸)组成的氨基酸序列。
在第七方面的一个实施方案中，切割产生切割产物A-Lys-Thr-Arg-Arg。在第七方面的进一步的实施方案中，切割产生切割产物A-Lys-Thr-Arg-Arg，产率为至少80％(例如，至少81％、至少82％、至少83％、至少84％、至少85％、至少86％、至少87％、至少88％、至少89％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％)。
在第八方面，本发明涉及组合物，其包含本发明的猪胰蛋白酶变体或本发明的核酸分子或本发明的宿主细胞。
在第八方面的一个实施方案中，组合物还包含赋形剂和/或至少一种其他活性剂。
在第八方面的一些实施方案中，组合物是药物组合物。
在第八方面的一些实施方案中，组合物或药物组合物是冻干的。
本发明还提供了本发明的猪胰蛋白酶变体(或本发明的核酸分子或本发明的宿主细胞)与至少一种其他活性药物成分的组合。在一个实施方案中，本发明的猪胰蛋白酶变体与至少一种其他活性药物成分的组合可以通过将活性药物成分单独向患者给予或以其中多种活性药物成分存在于一种药物组合物中的组合产物的形式来应用。在单独给予时，给予可以同时或以任何顺序按顺序进行。将选择本发明的活性剂和其他一种或多种活性药物成分的量和给予的相对时间安排，以便获得所需的组合治疗效果。组合的给予能以以下形式并行进行：(1)单一药物组合物，包括所有活性药物成分；或(2)单独的药物组合物，各自包括至少一种活性药物成分。可替代地，组合可以按顺序方式单独给予，其中首先给予一种治疗剂，之后给予另一种治疗剂，或反之亦然。这种按顺序给予可以在时间上相隔较近或在时间上相隔较远。
实施例
提出以下实施例以向本领域普通技术人员提供关于如何制备和使用本发明的化合物、组合物和方法的完整公开内容和描述，并不旨在以任何方式限制如随附权利要求所指示的本发明的范围。已经努力确保关于所使用的数字(例如，量、温度等)的准确性，但是应当考虑一些实验误差和偏差。除非另外指示，否则份数是重量份，分子量是平均分子量，温度是按摄氏度计，并且压力是大气压或接近大气压。
实施例1：编码猪胰蛋白酶的合成核苷酸序列的诱变和克隆
酶的改善可以通过酶工程化来实现。此技术涉及开发具有改善的特性的起始酶变体。为了提供酶变体，首先通过常规分子生物学方法在基因水平上在编码猪胰胰蛋白酶的合成核苷酸序列中引入突变。将酶变体的基因克隆到表达载体pLE1A17(pRSF-1b的衍生物，Novagen)中。所得质粒用于转化大肠杆菌(E.coli)BL21(DE3)细胞。
实施例2：变体猪胰蛋白酶原的表达
猪胰蛋白酶原在大肠杆菌中的重组表达在30℃下使用补充有卡那霉素(50mg/L)的培养基ZYM-5052通过根据F.W.Studier(F.W.Studier(2005)Prot.Exp.Pur.41:207-234)的自体诱导来进行。一旦培养物中的葡萄糖被代谢，存在的乳糖就诱导表达。使用甘油作为碳源。在微孔板以及在摇瓶规模上进行表达。
对于摇瓶实验，将补充有卡那霉素(50mg/L)和50μL甘油(36％)的20mL培养基ZYM-505的预培养物在30℃和200rpm下孵育24h。达到12.5–14的OD600。对于主要培养，使用500μL的预培养物接种100mL补充有卡那霉素(50mg/L)的培养基ZYM-5052。在30℃和200rpm下孵育24h之后，达到6.5–7.5的OD600。离心之后，将细胞沉淀物在-20℃下储存。
实施例3：细胞破坏和胰蛋白酶原活化
通过渗透休克释放周质胰蛋白酶原。将3.6g细胞(从实施例2获得)重悬在10mL裂解缓冲液(10mM Tris-HCl(pH＝8)、25mM MgCl2、200mM NaCl)中，直至获得均质悬浮液。将悬浮液在5℃和180rpm下搅拌24h。离心之后，对上清液进行活化。
对于胰蛋白酶原的活化，使用来自猪肠的肠激酶(Sigma，产品编号E0885；40个单位；单位定义：在25℃下，在pH 5.6下，一个单位的肠激酶将由胰蛋白酶原每分钟产生1.0纳摩尔胰蛋白酶)。
将肠激酶添加到无细胞提取物中，达到17mU/mL的最终浓度。将反应在30℃下孵育16–24h。将所得胰蛋白酶溶液在-20℃下储存。
实施例4：确定经纯化的变体重组猪胰蛋白酶的胰蛋白酶活性的测定在25℃下使用在100mM Tris pH 8.0、20mM CaCl2中的Chromozym TRY(Roche Diagnostics GmbH)确定胰蛋白酶的活性。Chromozym TRY是用于可靠地光度确定水解精氨酸的羧基侧的肽的蛋白酶(胰蛋白酶、内切蛋白酶Arg-C等)的活性的底物(苄氧羰基-L-缬氨酰基-L-甘氨酰基-L-精氨酸-4-苯基重氮酸乙酸酯)。在405nm下进行光度测量。
实施例5：人前胰岛素原的筛选结果。
使用人前胰岛素原(PPI)作为模型系统来进行所有胰蛋白酶变体的初步筛选。将PPI溶解在缓冲溶液(pH＝8.3)中，直到0.75g/L的浓度。根据实施例3制备胰蛋白酶变体溶液，并且无需进一步处理即添加到反应混合物中。定期取样，用1N HCl酸化并通过HPLC分析(实施例7)。
考虑了两个方面：(1)胰蛋白酶变体是否显示任何PPI切割活性(结果在表1，A列中示出)；(2)胰蛋白酶变体是否显示PPI切割活性：与SEQ ID NO:2相比，胰蛋白酶的切割选择性是否得到了改善(结果在表1，B列中示出)。为了评估胰蛋白酶变体的di-Arg选择性，通过HPLC监测B31Arg-B32Arg-人胰岛素的形成(参见实施例7)，并且与SEQ ID NO:2进行比较。根据以下计算选择性：
Figure BDA0002726189860000221
其中
[diArg]＝B31Arg-B32Arg-人胰岛素的浓度
[monoArg]＝desB32Arg-人胰岛素的浓度
[desThr]＝desB30Thr-人胰岛素的浓度
[UI]＝未知切割中间体的浓度
表1-人前胰岛素原的筛选结果
Figure BDA0002726189860000231
Figure BDA0002726189860000241
Figure BDA0002726189860000251
Figure BDA0002726189860000261
实施例6：使用重组SEQ ID NO:2(胰蛋白酶变体S172A)及其经优化的变体切割甘精前胰岛素原
这些实验在20℃和8.3的pH值(缓冲溶液)下进行，并且进行直到1000mL的规模。
将甘精前胰岛素原溶液填充在适当的恒温反应容器中，并且通过添加猪胰蛋白酶变体的溶液开始反应。在确定的时间间隔之后取样；通过使用1N或2N HCl溶液酸化样品溶液来立即停止酶促反应。通过HPLC分析样品(参见实施例7)。
图2示出了SEQ ID NO:2的反应动力学。所述图示出了甘精胰岛素的形成以及最主要的切割副产物B31Arg-甘精胰岛素(甘精胰岛素B链的位置31之后的切割，被称为“des-Arg”)和desB30Thr甘精胰岛素(甘精胰岛素B链的位置29之后的切割，被称为“des-Thr”)的形成的时间依赖性过程。
对所选的变体进行甘精前胰岛素原的切割。结果在表2中描绘。甘精胰岛素、desB32Arg-甘精胰岛素和desB29Thr-甘精胰岛素的含量以在切割结束时分析型HPLC色谱图的峰面积的形式给出，并且代表甘精胰岛素与副产物的比率。此外，摩尔切割产率根据以下给出：
Figure BDA0002726189860000262
其中
n(甘精胰岛素)＝形成的甘精胰岛素的摩尔数
n(甘精前胰岛素原)＝部署的甘精前胰岛素原的摩尔数
图4示出了作为经优化的胰蛋白酶变体的例子的猪胰蛋白酶变体105号(SEQ IDNO:105)的反应动力学。
表2–甘精前胰岛素原的切割结果的比较
Figure BDA0002726189860000271
Figure BDA0002726189860000281
如从表2可以清楚地看出，考虑B31Arg-甘精胰岛素(desArg)和desB30Thr-甘精胰岛素(desThr)，经优化的胰蛋白酶变体显示对甘精前胰岛素原的增强的切割选择性，从而导致产率显著增加，同时杂质分布降低。表1和表2的比较显示用人前胰岛素原和甘精前胰岛素原获得的切割结果的非常高的可比性。
实施例7：HPLC方法
Figure BDA0002726189860000282
Figure BDA0002726189860000291
流动相A 45mM磷酸钠缓冲液(pH 2.5)。315mM NaCl。25％(v/v)乙腈
流动相B 45mM磷酸钠缓冲液(pH 2.5)。55mM NaCl。65％(v/v)乙腈
Figure BDA0002726189860000292
进样体积：5μL
检测：UV(215nm)
实施例8：外肽酶活性
这些实验在8℃和8.3的pH值(缓冲溶液)下进行。
将甘精胰岛素溶液(0.5g/L)填充到适当的恒温反应容器中，并且通过添加SEQ IDNO:2溶液(100U/g胰岛素)开始反应。在确定的时间间隔之后取样，并且通过使用1N HCl溶液酸化样品溶液来立即停止酶促反应。通过HPLC分析样品(参见实施例7)。反应动力学在图3中描绘。
如可以在图3中清楚地看到，甘精胰岛素的浓度随时间降低。除形成少量的B30-desThr-甘精胰岛素以外，在此反应中形成的主要产物是B32-desArg-甘精胰岛素，表明胰蛋白酶SEQ ID NO:2能够通过外肽酶活性在位置B32处切掉单个精氨酸。
表3：胰蛋白酶变体的列表
Figure BDA0002726189860000293
Figure BDA0002726189860000301
Figure BDA0002726189860000311
Figure BDA0002726189860000321
Figure BDA0002726189860000331
野生型猪胰蛋白酶和所选的胰蛋白酶变体的全长序列：
野生型猪胰蛋白酶，SEQ ID NO:1：
Figure BDA0002726189860000332
变体S172A，SEQ ID NO:2：
Figure BDA0002726189860000333
在氨基酸位置24、44、56、78、99、107、125、131、170、172和193处具有可变位置的猪胰蛋白酶的通用序列；SEQ ID NO:3：
Figure BDA0002726189860000334
SEQ ID NO:4：
Figure BDA0002726189860000335
SEQ ID NO:5：
Figure BDA0002726189860000336
SEQ ID NO:6：
Figure BDA0002726189860000341
SEQ ID NO:7：
Figure BDA0002726189860000342
SEQ ID NO:8：
Figure BDA0002726189860000343
SEQ ID NO:9：
Figure BDA0002726189860000344
SEQ ID NO:10：
Figure BDA0002726189860000345
SEQ ID NO:11：
Figure BDA0002726189860000346
SEQ ID NO:12：
Figure BDA0002726189860000347
SEQ ID NO:13：
Figure BDA0002726189860000351
SEQ ID NO:14：
Figure BDA0002726189860000352
SEQ ID NO:15：
Figure BDA0002726189860000353
SEQ ID NO:16：
Figure BDA0002726189860000354
SEQ ID NO:17：
Figure BDA0002726189860000355
SEQ ID NO:18：
Figure BDA0002726189860000356
SEQ ID NO:19：
Figure BDA0002726189860000357
SEQ ID NO:20：
Figure BDA0002726189860000358
Figure BDA0002726189860000361
SEQ ID NO:21：
Figure BDA0002726189860000362
SEQ ID NO:22：
Figure BDA0002726189860000363
SEQ ID NO:23：
Figure BDA0002726189860000364
SEQ ID NO:24：
Figure BDA0002726189860000365
SEQ ID NO:25：
Figure BDA0002726189860000366
SEQ ID NO:26：
Figure BDA0002726189860000367
SEQ ID NO:27：
Figure BDA0002726189860000368
Figure BDA0002726189860000371
SEQ ID NO:28：
Figure BDA0002726189860000372
SEQ ID NO:29：
Figure BDA0002726189860000373
SEQ ID NO:30：
Figure BDA0002726189860000374
SEQ ID NO:31：
Figure BDA0002726189860000375
SEQ ID NO:32：
Figure BDA0002726189860000376
SEQ ID NO:33：
Figure BDA0002726189860000377
SEQ ID NO:34：
Figure BDA0002726189860000378
Figure BDA0002726189860000381
SEQ ID NO:35：
Figure BDA0002726189860000382
SEQ ID NO:36：
Figure BDA0002726189860000383
SEQ ID NO:37：
Figure BDA0002726189860000384
SEQ ID NO:38：
Figure BDA0002726189860000385
SEQ ID NO:39：
Figure BDA0002726189860000386
SEQ ID NO:40：
Figure BDA0002726189860000387
SEQ ID NO:41：
Figure BDA0002726189860000388
SEQ ID NO:42：
Figure BDA0002726189860000391
SEQ ID NO:43：
Figure BDA0002726189860000392
SEQ ID NO:44：
Figure BDA0002726189860000393
SEQ ID NO:45：
Figure BDA0002726189860000394
SEQ ID NO:46：
Figure BDA0002726189860000395
SEQ ID NO:47：
Figure BDA0002726189860000396
SEQ ID NO:48：
Figure BDA0002726189860000397
SEQ ID NO:49：
Figure BDA0002726189860000401
SEQ ID NO:50：
Figure BDA0002726189860000402
SEQ ID NO:51：
Figure BDA0002726189860000403
SEQ ID NO:52：
Figure BDA0002726189860000404
SEQ ID NO:53：
Figure BDA0002726189860000405
SEQ ID NO:54：
Figure BDA0002726189860000406
SEQ ID NO:55：
Figure BDA0002726189860000407
SEQ ID NO:56：
Figure BDA0002726189860000411
SEQ ID NO:57：
Figure BDA0002726189860000412
SEQ ID NO:58：
Figure BDA0002726189860000413
SEQ ID NO:59：
Figure BDA0002726189860000414
SEQ ID NO:60：
Figure BDA0002726189860000415
SEQ ID NO:61：
Figure BDA0002726189860000416
SEQ ID NO:62：
Figure BDA0002726189860000417
SEQ ID NO:63：
Figure BDA0002726189860000418
Figure BDA0002726189860000421
SEQ ID NO:64：
Figure BDA0002726189860000422
SEQ ID NO:65：
Figure BDA0002726189860000423
SEQ ID NO:66：
Figure BDA0002726189860000424
SEQ ID NO:67：
Figure BDA0002726189860000425
SEQ ID NO:68：
Figure BDA0002726189860000426
SEQ ID NO:69：
Figure BDA0002726189860000427
SEQ ID NO:70：
Figure BDA0002726189860000428
Figure BDA0002726189860000431
SEQ ID NO:71：
Figure BDA0002726189860000432
SEQ ID NO:72：
Figure BDA0002726189860000433
SEQ ID NO:73：
Figure BDA0002726189860000434
SEQ ID NO:74：
Figure BDA0002726189860000435
SEQ ID NO:75：
Figure BDA0002726189860000436
SEQ ID NO:76：
Figure BDA0002726189860000437
SEQ ID NO:77：
Figure BDA0002726189860000438
Figure BDA0002726189860000441
SEQ ID NO:78：
Figure BDA0002726189860000442
SEQ ID NO:79：
Figure BDA0002726189860000443
SEQ ID NO:80：
Figure BDA0002726189860000444
SEQ ID NO:81：
Figure BDA0002726189860000445
SEQ ID NO:82：
Figure BDA0002726189860000446
SEQ ID NO:83：
Figure BDA0002726189860000447
SEQ ID NO:84：
Figure BDA0002726189860000448
SEQ ID NO:85：
Figure BDA0002726189860000451
SEQ ID NO:86：
Figure BDA0002726189860000452
SEQ ID NO:87：
Figure BDA0002726189860000453
SEQ ID NO:88：
Figure BDA0002726189860000454
SEQ ID NO:89：
Figure BDA0002726189860000455
SEQ ID NO:90：
Figure BDA0002726189860000456
SEQ ID NO:91：
Figure BDA0002726189860000457
SEQ ID NO:92：
Figure BDA0002726189860000461
SEQ ID NO:93：
Figure BDA0002726189860000462
SEQ ID NO:94：
Figure BDA0002726189860000463
SEQ ID NO:95：
Figure BDA0002726189860000464
SEQ ID NO:96：
Figure BDA0002726189860000465
SEQ ID NO:97：
Figure BDA0002726189860000466
SEQ ID NO:98：
Figure BDA0002726189860000467
SEQ ID NO:99：
Figure BDA0002726189860000471
SEQ ID NO:100：
Figure BDA0002726189860000472
SEQ ID NO:101：
Figure BDA0002726189860000473
SEQ ID NO:102：
Figure BDA0002726189860000474
SEQ ID NO:103：
Figure BDA0002726189860000475
SEQ ID NO:104：
Figure BDA0002726189860000476
SEQ ID NO:105：
Figure BDA0002726189860000477
SEQ ID NO:106：
Figure BDA0002726189860000478
Figure BDA0002726189860000481
SEQ ID NO:107：
Figure BDA0002726189860000482
SEQ ID NO:108：
Figure BDA0002726189860000483
SEQ ID NO:109：
Figure BDA0002726189860000484
SEQ ID NO:110：
Figure BDA0002726189860000485
SEQ ID NO:111：
Figure BDA0002726189860000486
SEQ ID NO:112：
Figure BDA0002726189860000487
SEQ ID NO:113：
Figure BDA0002726189860000488
Figure BDA0002726189860000491
SEQ ID NO:114：
Figure BDA0002726189860000492
SEQ ID NO:115：
Figure BDA0002726189860000493
SEQ ID NO:116：
Figure BDA0002726189860000494
SEQ ID NO:117：
Figure BDA0002726189860000495
SEQ ID NO:118：
Figure BDA0002726189860000496
SEQ ID NO:119：
Figure BDA0002726189860000497
序列表
<110> 赛诺菲(SANOFI)
<120> 猪胰蛋白酶变体
<130> 589-276 PCT
<150> EP18158034.1
<151> 2018-02-22
<160> 130
<170> PatentIn 3.5版
<210> 1
<211> 223
<212> PRT
<213> 野猪(Sus scrofa)
<400> 1
Ile Val Gly Gly Tyr Thr Cys Ala Ala Asn Ser Ile Pro Tyr Gln Val
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Ser Leu Asn Ser Gly Ser His Phe Cys Gly Gly Ser Leu Ile Asn Ser
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Gln Trp Val Val Ser Ala Ala His Cys Tyr Lys Ser Arg Ile Gln Val
35 40 45
Arg Leu Gly Glu His Asn Ile Asp Val Leu Glu Gly Asn Glu Gln Phe
50 55 60
Ile Asn Ala Ala Lys Ile Ile Thr His Pro Asn Phe Asn Gly Asn Thr
65 70 75 80
Leu Asp Asn Asp Ile Met Leu Ile Lys Leu Ser Ser Pro Ala Thr Leu
85 90 95
Asn Ser Arg Val Ala Thr Val Ser Leu Pro Arg Ser Cys Ala Ala Ala
100 105 110
Gly Thr Glu Cys Leu Ile Ser Gly Trp Gly Asn Thr Lys Ser Ser Gly
115 120 125
Ser Ser Tyr Pro Ser Leu Leu Gln Cys Leu Lys Ala Pro Val Leu Ser
130 135 140
Asp Ser Ser Cys Lys Ser Ser Tyr Pro Gly Gln Ile Thr Gly Asn Met
145 150 155 160
Ile Cys Val Gly Phe Leu Glu Gly Gly Lys Asp Ser Cys Gln Gly Asp
165 170 175
Ser Gly Gly Pro Val Val Cys Asn Gly Gln Leu Gln Gly Ile Val Ser
180 185 190
Trp Gly Tyr Gly Cys Ala Gln Lys Asn Lys Pro Gly Val Tyr Thr Lys
195 200 205
Val Cys Asn Tyr Val Asn Trp Ile Gln Gln Thr Ile Ala Ala Asn
210 215 220
<210> 2
<211> 223
<212> PRT
<213> 人工序列
<220>
<223> 猪胰蛋白酶变体S172A
<400> 2
Ile Val Gly Gly Tyr Thr Cys Ala Ala Asn Ser Ile Pro Tyr Gln Val
1 5 10 15
Ser Leu Asn Ser Gly Ser His Phe Cys Gly Gly Ser Leu Ile Asn Ser
20 25 30
Gln Trp Val Val Ser Ala Ala His Cys Tyr Lys Ser Arg Ile Gln Val
35 40 45
Arg Leu Gly Glu His Asn Ile Asp Val Leu Glu Gly Asn Glu Gln Phe
50 55 60
Ile Asn Ala Ala Lys Ile Ile Thr His Pro Asn Phe Asn Gly Asn Thr
65 70 75 80
Leu Asp Asn Asp Ile Met Leu Ile Lys Leu Ser Ser Pro Ala Thr Leu
85 90 95
Asn Ser Arg Val Ala Thr Val Ser Leu Pro Arg Ser Cys Ala Ala Ala
100 105 110
Gly Thr Glu Cys Leu Ile Ser Gly Trp Gly Asn Thr Lys Ser Ser Gly
115 120 125
Ser Ser Tyr Pro Ser Leu Leu Gln Cys Leu Lys Ala Pro Val Leu Ser
130 135 140
Asp Ser Ser Cys Lys Ser Ser Tyr Pro Gly Gln Ile Thr Gly Asn Met
145 150 155 160
Ile Cys Val Gly Phe Leu Glu Gly Gly Lys Asp Ala Cys Gln Gly Asp
165 170 175
Ser Gly Gly Pro Val Val Cys Asn Gly Gln Leu Gln Gly Ile Val Ser
180 185 190
Trp Gly Tyr Gly Cys Ala Gln Lys Asn Lys Pro Gly Val Tyr Thr Lys
195 200 205
Val Cys Asn Tyr Val Asn Trp Ile Gln Gln Thr Ile Ala Ala Asn
210 215 220
<210> 3
<211> 223
<212> PRT
<213> 人工序列
<220>
<223> 在氨基酸位置24、44、56、78、99、107、125、131、170、172和193处具有可变位置的猪胰蛋白酶的通用序列
<220>
<221> 尚未归类的特征
<222> (24)..(24)
<223> Xaa选自Ala、Asn、Arg、Gln、Ile、Leu、Lys、Met、Phe、Ser、Thr和Val
<220>
<221> 尚未归类的特征
<222> (44)..(44)
<223> Xaa选自Leu、Pro和Ser
<220>
<221> 尚未归类的特征
<222> (56)..(56)
<223> Xaa选自Ala、Asn、Asp、His和Trp
<220>
<221> 尚未归类的特征
<222> (78)..(78)
<223> Xaa选自Ala、Glu、Gly、Pro、Ser和Tyr
<220>
<221> 尚未归类的特征
<222> (99)..(99)
<223> Xaa选自Ala、Arg、Asn、Asp、Glu、Gly、His、Leu、Phe、Thr、Trp和Tyr
<220>
<221> 尚未归类的特征
<222> (107)..(107)
<223> Xaa选自Arg、Asp、Gly、Pro、Ser和Thr
<220>
<221> 尚未归类的特征
<222> (125)..(125)
<223> Xaa选自Ala、Cys、Gln、Glu、Gly、His、Leu、Lys、Ser和Tyr
<220>
<221> 尚未归类的特征
<222> (131)..(131)
<223> Xaa选自Ala、Asn、Asp、Cys、Gln、Glu、Gly、His、Ile、Leu、Met、Ser、Thr、Trp、Tyr和Val
<220>
<221> 尚未归类的特征
<222> (170)..(170)
<223> Xaa选自Ala、Asn、Gly、Lys和Tyr
<220>
<221> 尚未归类的特征
<222> (172)..(172)
<223> Xaa选自Ala、Cys、Ser和Thr
<220>
<221> 尚未归类的特征
<222> (193)..(193)
<223> Xaa选自Phe、Ser、Thr、Trp和Tyr
<400> 3
Ile Val Gly Gly Tyr Thr Cys Ala Ala Asn Ser Ile Pro Tyr Gln Val
1 5 10 15
Ser Leu Asn Ser Gly Ser His Xaa Cys Gly Gly Ser Leu Ile Asn Ser
20 25 30
Gln Trp Val Val Ser Ala Ala His Cys Tyr Lys Xaa Arg Ile Gln Val
35 40 45
Arg Leu Gly Glu His Asn Ile Xaa Val Leu Glu Gly Asn Glu Gln Phe
50 55 60
Ile Asn Ala Ala Lys Ile Ile Thr His Pro Asn Phe Asn Xaa Asn Thr
65 70 75 80
Leu Asp Asn Asp Ile Met Leu Ile Lys Leu Ser Ser Pro Ala Thr Leu
85 90 95
Asn Ser Xaa Val Ala Thr Val Ser Leu Pro Xaa Ser Cys Ala Ala Ala
100 105 110
Gly Thr Glu Cys Leu Ile Ser Gly Trp Gly Asn Thr Xaa Ser Ser Gly
115 120 125
Ser Ser Xaa Pro Ser Leu Leu Gln Cys Leu Lys Ala Pro Val Leu Ser
130 135 140
Asp Ser Ser Cys Lys Ser Ser Tyr Pro Gly Gln Ile Thr Gly Asn Met
145 150 155 160
Ile Cys Val Gly Phe Leu Glu Gly Gly Xaa Asp Xaa Cys Gln Gly Asp
165 170 175
Ser Gly Gly Pro Val Val Cys Asn Gly Gln Leu Gln Gly Ile Val Ser
180 185 190
Xaa Gly Tyr Gly Cys Ala Gln Lys Asn Lys Pro Gly Val Tyr Thr Lys
195 200 205
Val Cys Asn Tyr Val Asn Trp Ile Gln Gln Thr Ile Ala Ala Asn
210 215 220
<210> 4
<211> 223
<212> PRT
<213> 人工序列
<220>
<223> 猪胰蛋白酶变体4号
<400> 4
Ile Val Gly Gly Tyr Thr Cys Ala Ala Asn Ser Ile Pro Tyr Gln Val
1 5 10 15
Ser Leu Asn Ser Gly Ser His Arg Cys Gly Gly Ser Leu Ile Asn Ser
20 25 30
Gln Trp Val Val Ser Ala Ala His Cys Tyr Lys Ser Arg Ile Gln Val
35 40 45
Arg Leu Gly Glu His Asn Ile Asp Val Leu Glu Gly Asn Glu Gln Phe
50 55 60
Ile Asn Ala Ala Lys Ile Ile Thr His Pro Asn Phe Asn Gly Asn Thr
65 70 75 80
Leu Asp Asn Asp Ile Met Leu Ile Lys Leu Ser Ser Pro Ala Thr Leu
85 90 95
Asn Ser Ala Val Ala Thr Val Ser Leu Pro Arg Ser Cys Ala Ala Ala
100 105 110
Gly Thr Glu Cys Leu Ile Ser Gly Trp Gly Asn Thr Lys Ser Ser Gly
115 120 125
Ser Ser Tyr Pro Ser Leu Leu Gln Cys Leu Lys Ala Pro Val Leu Ser
130 135 140
Asp Ser Ser Cys Lys Ser Ser Tyr Pro Gly Gln Ile Thr Gly Asn Met
145 150 155 160
Ile Cys Val Gly Phe Leu Glu Gly Gly Lys Asp Ala Cys Gln Gly Asp
165 170 175
Ser Gly Gly Pro Val Val Cys Asn Gly Gln Leu Gln Gly Ile Val Ser
180 185 190
Ser Gly Tyr Gly Cys Ala Gln Lys Asn Lys Pro Gly Val Tyr Thr Lys
195 200 205
Val Cys Asn Tyr Val Asn Trp Ile Gln Gln Thr Ile Ala Ala Asn
210 215 220
<210> 5
<211> 223
<212> PRT
<213> 人工序列
<220>
<223> 猪胰蛋白酶变体5号
<400> 5
Ile Val Gly Gly Tyr Thr Cys Ala Ala Asn Ser Ile Pro Tyr Gln Val
1 5 10 15
Ser Leu Asn Ser Gly Ser His Arg Cys Gly Gly Ser Leu Ile Asn Ser
20 25 30
Gln Trp Val Val Ser Ala Ala His Cys Tyr Lys Ser Arg Ile Gln Val
35 40 45
Arg Leu Gly Glu His Asn Ile Asp Val Leu Glu Gly Asn Glu Gln Phe
50 55 60
Ile Asn Ala Ala Lys Ile Ile Thr His Pro Asn Phe Asn Gly Asn Thr
65 70 75 80
Leu Asp Asn Asp Ile Met Leu Ile Lys Leu Ser Ser Pro Ala Thr Leu
85 90 95
Asn Ser Leu Val Ala Thr Val Ser Leu Pro Arg Ser Cys Ala Ala Ala
100 105 110
Gly Thr Glu Cys Leu Ile Ser Gly Trp Gly Asn Thr Lys Ser Ser Gly
115 120 125
Ser Ser Tyr Pro Ser Leu Leu Gln Cys Leu Lys Ala Pro Val Leu Ser
130 135 140
Asp Ser Ser Cys Lys Ser Ser Tyr Pro Gly Gln Ile Thr Gly Asn Met
145 150 155 160
Ile Cys Val Gly Phe Leu Glu Gly Gly Lys Asp Ala Cys Gln Gly Asp
165 170 175
Ser Gly Gly Pro Val Val Cys Asn Gly Gln Leu Gln Gly Ile Val Ser
180 185 190
Ser Gly Tyr Gly Cys Ala Gln Lys Asn Lys Pro Gly Val Tyr Thr Lys
195 200 205
Val Cys Asn Tyr Val Asn Trp Ile Gln Gln Thr Ile Ala Ala Asn
210 215 220
<210> 6
<211> 223
<212> PRT
<213> 人工序列
<220>
<223> 猪胰蛋白酶变体6号
<400> 6
Ile Val Gly Gly Tyr Thr Cys Ala Ala Asn Ser Ile Pro Tyr Gln Val
1 5 10 15
Ser Leu Asn Ser Gly Ser His Arg Cys Gly Gly Ser Leu Ile Asn Ser
20 25 30
Gln Trp Val Val Ser Ala Ala His Cys Tyr Lys Ser Arg Ile Gln Val
35 40 45
Arg Leu Gly Glu His Asn Ile Asp Val Leu Glu Gly Asn Glu Gln Phe
50 55 60
Ile Asn Ala Ala Lys Ile Ile Thr His Pro Asn Phe Asn Gly Asn Thr
65 70 75 80
Leu Asp Asn Asp Ile Met Leu Ile Lys Leu Ser Ser Pro Ala Thr Leu
85 90 95
Asn Ser Phe Val Ala Thr Val Ser Leu Pro Arg Ser Cys Ala Ala Ala
100 105 110
Gly Thr Glu Cys Leu Ile Ser Gly Trp Gly Asn Thr Lys Ser Ser Gly
115 120 125
Ser Ser Tyr Pro Ser Leu Leu Gln Cys Leu Lys Ala Pro Val Leu Ser
130 135 140
Asp Ser Ser Cys Lys Ser Ser Tyr Pro Gly Gln Ile Thr Gly Asn Met
145 150 155 160
Ile Cys Val Gly Phe Leu Glu Gly Gly Lys Asp Ala Cys Gln Gly Asp
165 170 175
Ser Gly Gly Pro Val Val Cys Asn Gly Gln Leu Gln Gly Ile Val Ser
180 185 190
Ser Gly Tyr Gly Cys Ala Gln Lys Asn Lys Pro Gly Val Tyr Thr Lys
195 200 205
Val Cys Asn Tyr Val Asn Trp Ile Gln Gln Thr Ile Ala Ala Asn
210 215 220
<210> 7
<211> 223
<212> PRT
<213> 人工序列
<220>
<223> 猪胰蛋白酶变体7号
<400> 7
Ile Val Gly Gly Tyr Thr Cys Ala Ala Asn Ser Ile Pro Tyr Gln Val
1 5 10 15
Ser Leu Asn Ser Gly Ser His Arg Cys Gly Gly Ser Leu Ile Asn Ser
20 25 30
Gln Trp Val Val Ser Ala Ala His Cys Tyr Lys Ser Arg Ile Gln Val
35 40 45
Arg Leu Gly Glu His Asn Ile Asp Val Leu Glu Gly Asn Glu Gln Phe
50 55 60
Ile Asn Ala Ala Lys Ile Ile Thr His Pro Asn Phe Asn Gly Asn Thr
65 70 75 80
Leu Asp Asn Asp Ile Met Leu Ile Lys Leu Ser Ser Pro Ala Thr Leu
85 90 95
Asn Ser Trp Val Ala Thr Val Ser Leu Pro Arg Ser Cys Ala Ala Ala
100 105 110
Gly Thr Glu Cys Leu Ile Ser Gly Trp Gly Asn Thr Lys Ser Ser Gly
115 120 125
Ser Ser Tyr Pro Ser Leu Leu Gln Cys Leu Lys Ala Pro Val Leu Ser
130 135 140
Asp Ser Ser Cys Lys Ser Ser Tyr Pro Gly Gln Ile Thr Gly Asn Met
145 150 155 160
Ile Cys Val Gly Phe Leu Glu Gly Gly Lys Asp Ala Cys Gln Gly Asp
165 170 175
Ser Gly Gly Pro Val Val Cys Asn Gly Gln Leu Gln Gly Ile Val Ser
180 185 190
Ser Gly Tyr Gly Cys Ala Gln Lys Asn Lys Pro Gly Val Tyr Thr Lys
195 200 205
Val Cys Asn Tyr Val Asn Trp Ile Gln Gln Thr Ile Ala Ala Asn
210 215 220
<210> 8
<211> 223
<212> PRT
<213> 人工序列
<220>
<223> 猪胰蛋白酶变体8号
<400> 8
Ile Val Gly Gly Tyr Thr Cys Ala Ala Asn Ser Ile Pro Tyr Gln Val
1 5 10 15
Ser Leu Asn Ser Gly Ser His Arg Cys Gly Gly Ser Leu Ile Asn Ser
20 25 30
Gln Trp Val Val Ser Ala Ala His Cys Tyr Lys Ser Arg Ile Gln Val
35 40 45
Arg Leu Gly Glu His Asn Ile Asp Val Leu Glu Gly Asn Glu Gln Phe
50 55 60
Ile Asn Ala Ala Lys Ile Ile Thr His Pro Asn Phe Asn Gly Asn Thr
65 70 75 80
Leu Asp Asn Asp Ile Met Leu Ile Lys Leu Ser Ser Pro Ala Thr Leu
85 90 95
Asn Ser Gly Val Ala Thr Val Ser Leu Pro Arg Ser Cys Ala Ala Ala
100 105 110
Gly Thr Glu Cys Leu Ile Ser Gly Trp Gly Asn Thr Lys Ser Ser Gly
115 120 125
Ser Ser Tyr Pro Ser Leu Leu Gln Cys Leu Lys Ala Pro Val Leu Ser
130 135 140
Asp Ser Ser Cys Lys Ser Ser Tyr Pro Gly Gln Ile Thr Gly Asn Met
145 150 155 160
Ile Cys Val Gly Phe Leu Glu Gly Gly Lys Asp Ala Cys Gln Gly Asp
165 170 175
Ser Gly Gly Pro Val Val Cys Asn Gly Gln Leu Gln Gly Ile Val Ser
180 185 190
Ser Gly Tyr Gly Cys Ala Gln Lys Asn Lys Pro Gly Val Tyr Thr Lys
195 200 205
Val Cys Asn Tyr Val Asn Trp Ile Gln Gln Thr Ile Ala Ala Asn
210 215 220
<210> 9
<211> 223
<212> PRT
<213> 人工序列
<220>
<223> 猪胰蛋白酶变体9号
<400> 9
Ile Val Gly Gly Tyr Thr Cys Ala Ala Asn Ser Ile Pro Tyr Gln Val
1 5 10 15
Ser Leu Asn Ser Gly Ser His Arg Cys Gly Gly Ser Leu Ile Asn Ser
20 25 30
Gln Trp Val Val Ser Ala Ala His Cys Tyr Lys Ser Arg Ile Gln Val
35 40 45
Arg Leu Gly Glu His Asn Ile Asp Val Leu Glu Gly Asn Glu Gln Phe
50 55 60
Ile Asn Ala Ala Lys Ile Ile Thr His Pro Asn Phe Asn Gly Asn Thr
65 70 75 80
Leu Asp Asn Asp Ile Met Leu Ile Lys Leu Ser Ser Pro Ala Thr Leu
85 90 95
Asn Ser Tyr Val Ala Thr Val Ser Leu Pro Arg Ser Cys Ala Ala Ala
100 105 110
Gly Thr Glu Cys Leu Ile Ser Gly Trp Gly Asn Thr Lys Ser Ser Gly
115 120 125
Ser Ser Tyr Pro Ser Leu Leu Gln Cys Leu Lys Ala Pro Val Leu Ser
130 135 140
Asp Ser Ser Cys Lys Ser Ser Tyr Pro Gly Gln Ile Thr Gly Asn Met
145 150 155 160
Ile Cys Val Gly Phe Leu Glu Gly Gly Lys Asp Ala Cys Gln Gly Asp
165 170 175
Ser Gly Gly Pro Val Val Cys Asn Gly Gln Leu Gln Gly Ile Val Ser
180 185 190
Ser Gly Tyr Gly Cys Ala Gln Lys Asn Lys Pro Gly Val Tyr Thr Lys
195 200 205
Val Cys Asn Tyr Val Asn Trp Ile Gln Gln Thr Ile Ala Ala Asn
210 215 220
<210> 10
<211> 223
<212> PRT
<213> 人工序列
<220>
<223> 猪胰蛋白酶变体10号
<400> 10
Ile Val Gly Gly Tyr Thr Cys Ala Ala Asn Ser Ile Pro Tyr Gln Val
1 5 10 15
Ser Leu Asn Ser Gly Ser His Arg Cys Gly Gly Ser Leu Ile Asn Ser
20 25 30
Gln Trp Val Val Ser Ala Ala His Cys Tyr Lys Ser Arg Ile Gln Val
35 40 45
Arg Leu Gly Glu His Asn Ile Asp Val Leu Glu Gly Asn Glu Gln Phe
50 55 60
Ile Asn Ala Ala Lys Ile Ile Thr His Pro Asn Phe Asn Gly Asn Thr
65 70 75 80
Leu Asp Asn Asp Ile Met Leu Ile Lys Leu Ser Ser Pro Ala Thr Leu
85 90 95
Asn Ser Thr Val Ala Thr Val Ser Leu Pro Arg Ser Cys Ala Ala Ala
100 105 110
Gly Thr Glu Cys Leu Ile Ser Gly Trp Gly Asn Thr Lys Ser Ser Gly
115 120 125
Ser Ser Tyr Pro Ser Leu Leu Gln Cys Leu Lys Ala Pro Val Leu Ser
130 135 140
Asp Ser Ser Cys Lys Ser Ser Tyr Pro Gly Gln Ile Thr Gly Asn Met
145 150 155 160
Ile Cys Val Gly Phe Leu Glu Gly Gly Lys Asp Ala Cys Gln Gly Asp
165 170 175
Ser Gly Gly Pro Val Val Cys Asn Gly Gln Leu Gln Gly Ile Val Ser
180 185 190
Ser Gly Tyr Gly Cys Ala Gln Lys Asn Lys Pro Gly Val Tyr Thr Lys
195 200 205
Val Cys Asn Tyr Val Asn Trp Ile Gln Gln Thr Ile Ala Ala Asn
210 215 220
<210> 11
<211> 223
<212> PRT
<213> 人工序列
<220>
<223> 猪胰蛋白酶变体11号
<400> 11
Ile Val Gly Gly Tyr Thr Cys Ala Ala Asn Ser Ile Pro Tyr Gln Val
1 5 10 15
Ser Leu Asn Ser Gly Ser His Arg Cys Gly Gly Ser Leu Ile Asn Ser
20 25 30
Gln Trp Val Val Ser Ala Ala His Cys Tyr Lys Ser Arg Ile Gln Val
35 40 45
Arg Leu Gly Glu His Asn Ile Asp Val Leu Glu Gly Asn Glu Gln Phe
50 55 60
Ile Asn Ala Ala Lys Ile Ile Thr His Pro Asn Phe Asn Gly Asn Thr
65 70 75 80
Leu Asp Asn Asp Ile Met Leu Ile Lys Leu Ser Ser Pro Ala Thr Leu
85 90 95
Asn Ser Asn Val Ala Thr Val Ser Leu Pro Arg Ser Cys Ala Ala Ala
100 105 110
Gly Thr Glu Cys Leu Ile Ser Gly Trp Gly Asn Thr Lys Ser Ser Gly
115 120 125
Ser Ser Tyr Pro Ser Leu Leu Gln Cys Leu Lys Ala Pro Val Leu Ser
130 135 140
Asp Ser Ser Cys Lys Ser Ser Tyr Pro Gly Gln Ile Thr Gly Asn Met
145 150 155 160
Ile Cys Val Gly Phe Leu Glu Gly Gly Lys Asp Ala Cys Gln Gly Asp
165 170 175
Ser Gly Gly Pro Val Val Cys Asn Gly Gln Leu Gln Gly Ile Val Ser
180 185 190
Ser Gly Tyr Gly Cys Ala Gln Lys Asn Lys Pro Gly Val Tyr Thr Lys
195 200 205
Val Cys Asn Tyr Val Asn Trp Ile Gln Gln Thr Ile Ala Ala Asn
210 215 220
<210> 12
<211> 223
<212> PRT
<213> 人工序列
<220>
<223> 猪胰蛋白酶变体12号
<400> 12
Ile Val Gly Gly Tyr Thr Cys Ala Ala Asn Ser Ile Pro Tyr Gln Val
1 5 10 15
Ser Leu Asn Ser Gly Ser His Arg Cys Gly Gly Ser Leu Ile Asn Ser
20 25 30
Gln Trp Val Val Ser Ala Ala His Cys Tyr Lys Ser Arg Ile Gln Val
35 40 45
Arg Leu Gly Glu His Asn Ile Asp Val Leu Glu Gly Asn Glu Gln Phe
50 55 60
Ile Asn Ala Ala Lys Ile Ile Thr His Pro Asn Phe Asn Gly Asn Thr
65 70 75 80
Leu Asp Asn Asp Ile Met Leu Ile Lys Leu Ser Ser Pro Ala Thr Leu
85 90 95
Asn Ser Asp Val Ala Thr Val Ser Leu Pro Arg Ser Cys Ala Ala Ala
100 105 110
Gly Thr Glu Cys Leu Ile Ser Gly Trp Gly Asn Thr Lys Ser Ser Gly
115 120 125
Ser Ser Tyr Pro Ser Leu Leu Gln Cys Leu Lys Ala Pro Val Leu Ser
130 135 140
Asp Ser Ser Cys Lys Ser Ser Tyr Pro Gly Gln Ile Thr Gly Asn Met
145 150 155 160
Ile Cys Val Gly Phe Leu Glu Gly Gly Lys Asp Ala Cys Gln Gly Asp
165 170 175
Ser Gly Gly Pro Val Val Cys Asn Gly Gln Leu Gln Gly Ile Val Ser
180 185 190
Ser Gly Tyr Gly Cys Ala Gln Lys Asn Lys Pro Gly Val Tyr Thr Lys
195 200 205
Val Cys Asn Tyr Val Asn Trp Ile Gln Gln Thr Ile Ala Ala Asn
210 215 220
<210> 13
<211> 223
<212> PRT
<213> 人工序列
<220>
<223> 猪胰蛋白酶变体13号
<400> 13
Ile Val Gly Gly Tyr Thr Cys Ala Ala Asn Ser Ile Pro Tyr Gln Val
1 5 10 15
Ser Leu Asn Ser Gly Ser His Arg Cys Gly Gly Ser Leu Ile Asn Ser
20 25 30
Gln Trp Val Val Ser Ala Ala His Cys Tyr Lys Ser Arg Ile Gln Val
35 40 45
Arg Leu Gly Glu His Asn Ile Asp Val Leu Glu Gly Asn Glu Gln Phe
50 55 60
Ile Asn Ala Ala Lys Ile Ile Thr His Pro Asn Phe Asn Gly Asn Thr
65 70 75 80
Leu Asp Asn Asp Ile Met Leu Ile Lys Leu Ser Ser Pro Ala Thr Leu
85 90 95
Asn Ser Glu Val Ala Thr Val Ser Leu Pro Arg Ser Cys Ala Ala Ala
100 105 110
Gly Thr Glu Cys Leu Ile Ser Gly Trp Gly Asn Thr Lys Ser Ser Gly
115 120 125
Ser Ser Tyr Pro Ser Leu Leu Gln Cys Leu Lys Ala Pro Val Leu Ser
130 135 140
Asp Ser Ser Cys Lys Ser Ser Tyr Pro Gly Gln Ile Thr Gly Asn Met
145 150 155 160
Ile Cys Val Gly Phe Leu Glu Gly Gly Lys Asp Ala Cys Gln Gly Asp
165 170 175
Ser Gly Gly Pro Val Val Cys Asn Gly Gln Leu Gln Gly Ile Val Ser
180 185 190
Ser Gly Tyr Gly Cys Ala Gln Lys Asn Lys Pro Gly Val Tyr Thr Lys
195 200 205
Val Cys Asn Tyr Val Asn Trp Ile Gln Gln Thr Ile Ala Ala Asn
210 215 220
<210> 14
<211> 223
<212> PRT
<213> 人工序列
<220>
<223> 猪胰蛋白酶变体14号
<400> 14
Ile Val Gly Gly Tyr Thr Cys Ala Ala Asn Ser Ile Pro Tyr Gln Val
1 5 10 15
Ser Leu Asn Ser Gly Ser His Arg Cys Gly Gly Ser Leu Ile Asn Ser
20 25 30
Gln Trp Val Val Ser Ala Ala His Cys Tyr Lys Ser Arg Ile Gln Val
35 40 45
Arg Leu Gly Glu His Asn Ile Asp Val Leu Glu Gly Asn Glu Gln Phe
50 55 60
Ile Asn Ala Ala Lys Ile Ile Thr His Pro Asn Phe Asn Gly Asn Thr
65 70 75 80
Leu Asp Asn Asp Ile Met Leu Ile Lys Leu Ser Ser Pro Ala Thr Leu
85 90 95
Asn Ser His Val Ala Thr Val Ser Leu Pro Arg Ser Cys Ala Ala Ala
100 105 110
Gly Thr Glu Cys Leu Ile Ser Gly Trp Gly Asn Thr Lys Ser Ser Gly
115 120 125
Ser Ser Tyr Pro Ser Leu Leu Gln Cys Leu Lys Ala Pro Val Leu Ser
130 135 140
Asp Ser Ser Cys Lys Ser Ser Tyr Pro Gly Gln Ile Thr Gly Asn Met
145 150 155 160
Ile Cys Val Gly Phe Leu Glu Gly Gly Lys Asp Ala Cys Gln Gly Asp
165 170 175
Ser Gly Gly Pro Val Val Cys Asn Gly Gln Leu Gln Gly Ile Val Ser
180 185 190
Ser Gly Tyr Gly Cys Ala Gln Lys Asn Lys Pro Gly Val Tyr Thr Lys
195 200 205
Val Cys Asn Tyr Val Asn Trp Ile Gln Gln Thr Ile Ala Ala Asn
210 215 220
<210> 15
<211> 223
<212> PRT
<213> 人工序列
<220>
<223> 猪胰蛋白酶变体15号
<400> 15
Ile Val Gly Gly Tyr Thr Cys Ala Ala Asn Ser Ile Pro Tyr Gln Val
1 5 10 15
Ser Leu Asn Ser Gly Ser His Arg Cys Gly Gly Ser Leu Ile Asn Ser
20 25 30
Gln Trp Val Val Ser Ala Ala His Cys Tyr Lys Ser Arg Ile Gln Val
35 40 45
Arg Leu Gly Glu His Asn Ile Asp Val Leu Glu Gly Asn Glu Gln Phe
50 55 60
Ile Asn Ala Ala Lys Ile Ile Thr His Pro Asn Phe Asn Gly Asn Thr
65 70 75 80
Leu Asp Asn Asp Ile Met Leu Ile Lys Leu Ser Ser Pro Ala Thr Leu
85 90 95
Asn Ser Arg Val Ala Thr Val Ser Leu Pro Arg Ser Cys Ala Ala Ala
100 105 110
Gly Thr Glu Cys Leu Ile Ser Gly Trp Gly Asn Thr Ala Ser Ser Gly
115 120 125
Ser Ser Tyr Pro Ser Leu Leu Gln Cys Leu Lys Ala Pro Val Leu Ser
130 135 140
Asp Ser Ser Cys Lys Ser Ser Tyr Pro Gly Gln Ile Thr Gly Asn Met
145 150 155 160
Ile Cys Val Gly Phe Leu Glu Gly Gly Lys Asp Ala Cys Gln Gly Asp
165 170 175
Ser Gly Gly Pro Val Val Cys Asn Gly Gln Leu Gln Gly Ile Val Ser
180 185 190
Ser Gly Tyr Gly Cys Ala Gln Lys Asn Lys Pro Gly Val Tyr Thr Lys
195 200 205
Val Cys Asn Tyr Val Asn Trp Ile Gln Gln Thr Ile Ala Ala Asn
210 215 220
<210> 16
<211> 223
<212> PRT
<213> 人工序列
<220>
<223> 猪胰蛋白酶变体16号
<400> 16
Ile Val Gly Gly Tyr Thr Cys Ala Ala Asn Ser Ile Pro Tyr Gln Val
1 5 10 15
Ser Leu Asn Ser Gly Ser His Arg Cys Gly Gly Ser Leu Ile Asn Ser
20 25 30
Gln Trp Val Val Ser Ala Ala His Cys Tyr Lys Ser Arg Ile Gln Val
35 40 45
Arg Leu Gly Glu His Asn Ile Asp Val Leu Glu Gly Asn Glu Gln Phe
50 55 60
Ile Asn Ala Ala Lys Ile Ile Thr His Pro Asn Phe Asn Gly Asn Thr
65 70 75 80
Leu Asp Asn Asp Ile Met Leu Ile Lys Leu Ser Ser Pro Ala Thr Leu
85 90 95
Asn Ser Arg Val Ala Thr Val Ser Leu Pro Arg Ser Cys Ala Ala Ala
100 105 110
Gly Thr Glu Cys Leu Ile Ser Gly Trp Gly Asn Thr Gly Ser Ser Gly
115 120 125
Ser Ser Tyr Pro Ser Leu Leu Gln Cys Leu Lys Ala Pro Val Leu Ser
130 135 140
Asp Ser Ser Cys Lys Ser Ser Tyr Pro Gly Gln Ile Thr Gly Asn Met
145 150 155 160
Ile Cys Val Gly Phe Leu Glu Gly Gly Lys Asp Ala Cys Gln Gly Asp
165 170 175
Ser Gly Gly Pro Val Val Cys Asn Gly Gln Leu Gln Gly Ile Val Ser
180 185 190
Ser Gly Tyr Gly Cys Ala Gln Lys Asn Lys Pro Gly Val Tyr Thr Lys
195 200 205
Val Cys Asn Tyr Val Asn Trp Ile Gln Gln Thr Ile Ala Ala Asn
210 215 220
<210> 17
<211> 223
<212> PRT
<213> 人工序列
<220>
<223> 猪胰蛋白酶变体17号
<400> 17
Ile Val Gly Gly Tyr Thr Cys Ala Ala Asn Ser Ile Pro Tyr Gln Val
1 5 10 15
Ser Leu Asn Ser Gly Ser His Arg Cys Gly Gly Ser Leu Ile Asn Ser
20 25 30
Gln Trp Val Val Ser Ala Ala His Cys Tyr Lys Ser Arg Ile Gln Val
35 40 45
Arg Leu Gly Glu His Asn Ile Asp Val Leu Glu Gly Asn Glu Gln Phe
50 55 60
Ile Asn Ala Ala Lys Ile Ile Thr His Pro Asn Phe Asn Gly Asn Thr
65 70 75 80
Leu Asp Asn Asp Ile Met Leu Ile Lys Leu Ser Ser Pro Ala Thr Leu
85 90 95
Asn Ser Arg Val Ala Thr Val Ser Leu Pro Arg Ser Cys Ala Ala Ala
100 105 110
Gly Thr Glu Cys Leu Ile Ser Gly Trp Gly Asn Thr Tyr Ser Ser Gly
115 120 125
Ser Ser Tyr Pro Ser Leu Leu Gln Cys Leu Lys Ala Pro Val Leu Ser
130 135 140
Asp Ser Ser Cys Lys Ser Ser Tyr Pro Gly Gln Ile Thr Gly Asn Met
145 150 155 160
Ile Cys Val Gly Phe Leu Glu Gly Gly Lys Asp Ala Cys Gln Gly Asp
165 170 175
Ser Gly Gly Pro Val Val Cys Asn Gly Gln Leu Gln Gly Ile Val Ser
180 185 190
Ser Gly Tyr Gly Cys Ala Gln Lys Asn Lys Pro Gly Val Tyr Thr Lys
195 200 205
Val Cys Asn Tyr Val Asn Trp Ile Gln Gln Thr Ile Ala Ala Asn
210 215 220
<210> 18
<211> 223
<212> PRT
<213> 人工序列
<220>
<223> 猪胰蛋白酶变体18号
<400> 18
Ile Val Gly Gly Tyr Thr Cys Ala Ala Asn Ser Ile Pro Tyr Gln Val
1 5 10 15
Ser Leu Asn Ser Gly Ser His Arg Cys Gly Gly Ser Leu Ile Asn Ser
20 25 30
Gln Trp Val Val Ser Ala Ala His Cys Tyr Lys Ser Arg Ile Gln Val
35 40 45
Arg Leu Gly Glu His Asn Ile Asp Val Leu Glu Gly Asn Glu Gln Phe
50 55 60
Ile Asn Ala Ala Lys Ile Ile Thr His Pro Asn Phe Asn Gly Asn Thr
65 70 75 80
Leu Asp Asn Asp Ile Met Leu Ile Lys Leu Ser Ser Pro Ala Thr Leu
85 90 95
Asn Ser Arg Val Ala Thr Val Ser Leu Pro Arg Ser Cys Ala Ala Ala
100 105 110
Gly Thr Glu Cys Leu Ile Ser Gly Trp Gly Asn Thr Ser Ser Ser Gly
115 120 125
Ser Ser Tyr Pro Ser Leu Leu Gln Cys Leu Lys Ala Pro Val Leu Ser
130 135 140
Asp Ser Ser Cys Lys Ser Ser Tyr Pro Gly Gln Ile Thr Gly Asn Met
145 150 155 160
Ile Cys Val Gly Phe Leu Glu Gly Gly Lys Asp Ala Cys Gln Gly Asp
165 170 175
Ser Gly Gly Pro Val Val Cys Asn Gly Gln Leu Gln Gly Ile Val Ser
180 185 190
Ser Gly Tyr Gly Cys Ala Gln Lys Asn Lys Pro Gly Val Tyr Thr Lys
195 200 205
Val Cys Asn Tyr Val Asn Trp Ile Gln Gln Thr Ile Ala Ala Asn
210 215 220
<210> 19
<211> 223
<212> PRT
<213> 人工序列
<220>
<223> 猪胰蛋白酶变体19号
<400> 19
Ile Val Gly Gly Tyr Thr Cys Ala Ala Asn Ser Ile Pro Tyr Gln Val
1 5 10 15
Ser Leu Asn Ser Gly Ser His Arg Cys Gly Gly Ser Leu Ile Asn Ser
20 25 30
Gln Trp Val Val Ser Ala Ala His Cys Tyr Lys Ser Arg Ile Gln Val
35 40 45
Arg Leu Gly Glu His Asn Ile Asp Val Leu Glu Gly Asn Glu Gln Phe
50 55 60
Ile Asn Ala Ala Lys Ile Ile Thr His Pro Asn Phe Asn Gly Asn Thr
65 70 75 80
Leu Asp Asn Asp Ile Met Leu Ile Lys Leu Ser Ser Pro Ala Thr Leu
85 90 95
Asn Ser Arg Val Ala Thr Val Ser Leu Pro Arg Ser Cys Ala Ala Ala
100 105 110
Gly Thr Glu Cys Leu Ile Ser Gly Trp Gly Asn Thr Cys Ser Ser Gly
115 120 125
Ser Ser Tyr Pro Ser Leu Leu Gln Cys Leu Lys Ala Pro Val Leu Ser
130 135 140
Asp Ser Ser Cys Lys Ser Ser Tyr Pro Gly Gln Ile Thr Gly Asn Met
145 150 155 160
Ile Cys Val Gly Phe Leu Glu Gly Gly Lys Asp Ala Cys Gln Gly Asp
165 170 175
Ser Gly Gly Pro Val Val Cys Asn Gly Gln Leu Gln Gly Ile Val Ser
180 185 190
Ser Gly Tyr Gly Cys Ala Gln Lys Asn Lys Pro Gly Val Tyr Thr Lys
195 200 205
Val Cys Asn Tyr Val Asn Trp Ile Gln Gln Thr Ile Ala Ala Asn
210 215 220
<210> 20
<211> 223
<212> PRT
<213> 人工序列
<220>
<223> 猪胰蛋白酶变体20号
<400> 20
Ile Val Gly Gly Tyr Thr Cys Ala Ala Asn Ser Ile Pro Tyr Gln Val
1 5 10 15
Ser Leu Asn Ser Gly Ser His Arg Cys Gly Gly Ser Leu Ile Asn Ser
20 25 30
Gln Trp Val Val Ser Ala Ala His Cys Tyr Lys Ser Arg Ile Gln Val
35 40 45
Arg Leu Gly Glu His Asn Ile Asp Val Leu Glu Gly Asn Glu Gln Phe
50 55 60
Ile Asn Ala Ala Lys Ile Ile Thr His Pro Asn Phe Asn Gly Asn Thr
65 70 75 80
Leu Asp Asn Asp Ile Met Leu Ile Lys Leu Ser Ser Pro Ala Thr Leu
85 90 95
Asn Ser Arg Val Ala Thr Val Ser Leu Pro Arg Ser Cys Ala Ala Ala
100 105 110
Gly Thr Glu Cys Leu Ile Ser Gly Trp Gly Asn Thr Glu Ser Ser Gly
115 120 125
Ser Ser Tyr Pro Ser Leu Leu Gln Cys Leu Lys Ala Pro Val Leu Ser
130 135 140
Asp Ser Ser Cys Lys Ser Ser Tyr Pro Gly Gln Ile Thr Gly Asn Met
145 150 155 160
Ile Cys Val Gly Phe Leu Glu Gly Gly Lys Asp Ala Cys Gln Gly Asp
165 170 175
Ser Gly Gly Pro Val Val Cys Asn Gly Gln Leu Gln Gly Ile Val Ser
180 185 190
Ser Gly Tyr Gly Cys Ala Gln Lys Asn Lys Pro Gly Val Tyr Thr Lys
195 200 205
Val Cys Asn Tyr Val Asn Trp Ile Gln Gln Thr Ile Ala Ala Asn
210 215 220
<210> 21
<211> 223
<212> PRT
<213> 人工序列
<220>
<223> 猪胰蛋白酶变体21号
<400> 21
Ile Val Gly Gly Tyr Thr Cys Ala Ala Asn Ser Ile Pro Tyr Gln Val
1 5 10 15
Ser Leu Asn Ser Gly Ser His Arg Cys Gly Gly Ser Leu Ile Asn Ser
20 25 30
Gln Trp Val Val Ser Ala Ala His Cys Tyr Lys Ser Arg Ile Gln Val
35 40 45
Arg Leu Gly Glu His Asn Ile Asp Val Leu Glu Gly Asn Glu Gln Phe
50 55 60
Ile Asn Ala Ala Lys Ile Ile Thr His Pro Asn Phe Asn Gly Asn Thr
65 70 75 80
Leu Asp Asn Asp Ile Met Leu Ile Lys Leu Ser Ser Pro Ala Thr Leu
85 90 95
Asn Ser Arg Val Ala Thr Val Ser Leu Pro Arg Ser Cys Ala Ala Ala
100 105 110
Gly Thr Glu Cys Leu Ile Ser Gly Trp Gly Asn Thr His Ser Ser Gly
115 120 125
Ser Ser Tyr Pro Ser Leu Leu Gln Cys Leu Lys Ala Pro Val Leu Ser
130 135 140
Asp Ser Ser Cys Lys Ser Ser Tyr Pro Gly Gln Ile Thr Gly Asn Met
145 150 155 160
Ile Cys Val Gly Phe Leu Glu Gly Gly Lys Asp Ala Cys Gln Gly Asp
165 170 175
Ser Gly Gly Pro Val Val Cys Asn Gly Gln Leu Gln Gly Ile Val Ser
180 185 190
Ser Gly Tyr Gly Cys Ala Gln Lys Asn Lys Pro Gly Val Tyr Thr Lys
195 200 205
Val Cys Asn Tyr Val Asn Trp Ile Gln Gln Thr Ile Ala Ala Asn
210 215 220
<210> 22
<211> 223
<212> PRT
<213> 人工序列
<220>
<223> 猪胰蛋白酶变体22号
<400> 22
Ile Val Gly Gly Tyr Thr Cys Ala Ala Asn Ser Ile Pro Tyr Gln Val
1 5 10 15
Ser Leu Asn Ser Gly Ser His Arg Cys Gly Gly Ser Leu Ile Asn Ser
20 25 30
Gln Trp Val Val Ser Ala Ala His Cys Tyr Lys Ser Arg Ile Gln Val
35 40 45
Arg Leu Gly Glu His Asn Ile Asp Val Leu Glu Gly Asn Glu Gln Phe
50 55 60
Ile Asn Ala Ala Lys Ile Ile Thr His Pro Asn Phe Asn Gly Asn Thr
65 70 75 80
Leu Asp Asn Asp Ile Met Leu Ile Lys Leu Ser Ser Pro Ala Thr Leu
85 90 95
Asn Ser Arg Val Ala Thr Val Ser Leu Pro Arg Ser Cys Ala Ala Ala
100 105 110
Gly Thr Glu Cys Leu Ile Ser Gly Trp Gly Asn Thr Leu Ser Ser Gly
115 120 125
Ser Ser Tyr Pro Ser Leu Leu Gln Cys Leu Lys Ala Pro Val Leu Ser
130 135 140
Asp Ser Ser Cys Lys Ser Ser Tyr Pro Gly Gln Ile Thr Gly Asn Met
145 150 155 160
Ile Cys Val Gly Phe Leu Glu Gly Gly Lys Asp Ala Cys Gln Gly Asp
165 170 175
Ser Gly Gly Pro Val Val Cys Asn Gly Gln Leu Gln Gly Ile Val Ser
180 185 190
Ser Gly Tyr Gly Cys Ala Gln Lys Asn Lys Pro Gly Val Tyr Thr Lys
195 200 205
Val Cys Asn Tyr Val Asn Trp Ile Gln Gln Thr Ile Ala Ala Asn
210 215 220
<210> 23
<211> 223
<212> PRT
<213> 人工序列
<220>
<223> 猪胰蛋白酶变体23号
<400> 23
Ile Val Gly Gly Tyr Thr Cys Ala Ala Asn Ser Ile Pro Tyr Gln Val
1 5 10 15
Ser Leu Asn Ser Gly Ser His Arg Cys Gly Gly Ser Leu Ile Asn Ser
20 25 30
Gln Trp Val Val Ser Ala Ala His Cys Tyr Lys Ser Arg Ile Gln Val
35 40 45
Arg Leu Gly Glu His Asn Ile Asp Val Leu Glu Gly Asn Glu Gln Phe
50 55 60
Ile Asn Ala Ala Lys Ile Ile Thr His Pro Asn Phe Asn Gly Asn Thr
65 70 75 80
Leu Asp Asn Asp Ile Met Leu Ile Lys Leu Ser Ser Pro Ala Thr Leu
85 90 95
Asn Ser Arg Val Ala Thr Val Ser Leu Pro Arg Ser Cys Ala Ala Ala
100 105 110
Gly Thr Glu Cys Leu Ile Ser Gly Trp Gly Asn Thr Gln Ser Ser Gly
115 120 125
Ser Ser Tyr Pro Ser Leu Leu Gln Cys Leu Lys Ala Pro Val Leu Ser
130 135 140
Asp Ser Ser Cys Lys Ser Ser Tyr Pro Gly Gln Ile Thr Gly Asn Met
145 150 155 160
Ile Cys Val Gly Phe Leu Glu Gly Gly Lys Asp Ala Cys Gln Gly Asp
165 170 175
Ser Gly Gly Pro Val Val Cys Asn Gly Gln Leu Gln Gly Ile Val Ser
180 185 190
Ser Gly Tyr Gly Cys Ala Gln Lys Asn Lys Pro Gly Val Tyr Thr Lys
195 200 205
Val Cys Asn Tyr Val Asn Trp Ile Gln Gln Thr Ile Ala Ala Asn
210 215 220
<210> 24
<211> 223
<212> PRT
<213> 人工序列
<220>
<223> 猪胰蛋白酶变体24号
<400> 24
Ile Val Gly Gly Tyr Thr Cys Ala Ala Asn Ser Ile Pro Tyr Gln Val
1 5 10 15
Ser Leu Asn Ser Gly Ser His Arg Cys Gly Gly Ser Leu Ile Asn Ser
20 25 30
Gln Trp Val Val Ser Ala Ala His Cys Tyr Lys Ser Arg Ile Gln Val
35 40 45
Arg Leu Gly Glu His Asn Ile Asp Val Leu Glu Gly Asn Glu Gln Phe
50 55 60
Ile Asn Ala Ala Lys Ile Ile Thr His Pro Asn Phe Asn Gly Asn Thr
65 70 75 80
Leu Asp Asn Asp Ile Met Leu Ile Lys Leu Ser Ser Pro Ala Thr Leu
85 90 95
Asn Ser Arg Val Ala Thr Val Ser Leu Pro Arg Ser Cys Ala Ala Ala
100 105 110
Gly Thr Glu Cys Leu Ile Ser Gly Trp Gly Asn Thr Lys Ser Ser Gly
115 120 125
Ser Ser Tyr Pro Ser Leu Leu Gln Cys Leu Lys Ala Pro Val Leu Ser
130 135 140
Asp Ser Ser Cys Lys Ser Ser Tyr Pro Gly Gln Ile Thr Gly Asn Met
145 150 155 160
Ile Cys Val Gly Phe Leu Glu Gly Gly Tyr Asp Ala Cys Gln Gly Asp
165 170 175
Ser Gly Gly Pro Val Val Cys Asn Gly Gln Leu Gln Gly Ile Val Ser
180 185 190
Ser Gly Tyr Gly Cys Ala Gln Lys Asn Lys Pro Gly Val Tyr Thr Lys
195 200 205
Val Cys Asn Tyr Val Asn Trp Ile Gln Gln Thr Ile Ala Ala Asn
210 215 220
<210> 25
<211> 223
<212> PRT
<213> 人工序列
<220>
<223> 猪胰蛋白酶变体25号
<400> 25
Ile Val Gly Gly Tyr Thr Cys Ala Ala Asn Ser Ile Pro Tyr Gln Val
1 5 10 15
Ser Leu Asn Ser Gly Ser His Arg Cys Gly Gly Ser Leu Ile Asn Ser
20 25 30
Gln Trp Val Val Ser Ala Ala His Cys Tyr Lys Ser Arg Ile Gln Val
35 40 45
Arg Leu Gly Glu His Asn Ile Asp Val Leu Glu Gly Asn Glu Gln Phe
50 55 60
Ile Asn Ala Ala Lys Ile Ile Thr His Pro Asn Phe Asn Gly Asn Thr
65 70 75 80
Leu Asp Asn Asp Ile Met Leu Ile Lys Leu Ser Ser Pro Ala Thr Leu
85 90 95
Asn Ser Arg Val Ala Thr Val Ser Leu Pro Arg Ser Cys Ala Ala Ala
100 105 110
Gly Thr Glu Cys Leu Ile Ser Gly Trp Gly Asn Thr Lys Ser Ser Gly
115 120 125
Ser Ser Tyr Pro Ser Leu Leu Gln Cys Leu Lys Ala Pro Val Leu Ser
130 135 140
Asp Ser Ser Cys Lys Ser Ser Tyr Pro Gly Gln Ile Thr Gly Asn Met
145 150 155 160
Ile Cys Val Gly Phe Leu Glu Gly Gly Ala Asp Ala Cys Gln Gly Asp
165 170 175
Ser Gly Gly Pro Val Val Cys Asn Gly Gln Leu Gln Gly Ile Val Ser
180 185 190
Ser Gly Tyr Gly Cys Ala Gln Lys Asn Lys Pro Gly Val Tyr Thr Lys
195 200 205
Val Cys Asn Tyr Val Asn Trp Ile Gln Gln Thr Ile Ala Ala Asn
210 215 220
<210> 26
<211> 223
<212> PRT
<213> 人工序列
<220>
<223> 猪胰蛋白酶变体26号
<400> 26
Ile Val Gly Gly Tyr Thr Cys Ala Ala Asn Ser Ile Pro Tyr Gln Val
1 5 10 15
Ser Leu Asn Ser Gly Ser His Arg Cys Gly Gly Ser Leu Ile Asn Ser
20 25 30
Gln Trp Val Val Ser Ala Ala His Cys Tyr Lys Ser Arg Ile Gln Val
35 40 45
Arg Leu Gly Glu His Asn Ile Asp Val Leu Glu Gly Asn Glu Gln Phe
50 55 60
Ile Asn Ala Ala Lys Ile Ile Thr His Pro Asn Phe Asn Gly Asn Thr
65 70 75 80
Leu Asp Asn Asp Ile Met Leu Ile Lys Leu Ser Ser Pro Ala Thr Leu
85 90 95
Asn Ser Arg Val Ala Thr Val Ser Leu Pro Arg Ser Cys Ala Ala Ala
100 105 110
Gly Thr Glu Cys Leu Ile Ser Gly Trp Gly Asn Thr Lys Ser Ser Gly
115 120 125
Ser Ser Tyr Pro Ser Leu Leu Gln Cys Leu Lys Ala Pro Val Leu Ser
130 135 140
Asp Ser Ser Cys Lys Ser Ser Tyr Pro Gly Gln Ile Thr Gly Asn Met
145 150 155 160
Ile Cys Val Gly Phe Leu Glu Gly Gly Gly Asp Ala Cys Gln Gly Asp
165 170 175
Ser Gly Gly Pro Val Val Cys Asn Gly Gln Leu Gln Gly Ile Val Ser
180 185 190
Ser Gly Tyr Gly Cys Ala Gln Lys Asn Lys Pro Gly Val Tyr Thr Lys
195 200 205
Val Cys Asn Tyr Val Asn Trp Ile Gln Gln Thr Ile Ala Ala Asn
210 215 220
<210> 27
<211> 223
<212> PRT
<213> 人工序列
<220>
<223> 猪胰蛋白酶变体27号
<400> 27
Ile Val Gly Gly Tyr Thr Cys Ala Ala Asn Ser Ile Pro Tyr Gln Val
1 5 10 15
Ser Leu Asn Ser Gly Ser His Arg Cys Gly Gly Ser Leu Ile Asn Ser
20 25 30
Gln Trp Val Val Ser Ala Ala His Cys Tyr Lys Ser Arg Ile Gln Val
35 40 45
Arg Leu Gly Glu His Asn Ile Asp Val Leu Glu Gly Asn Glu Gln Phe
50 55 60
Ile Asn Ala Ala Lys Ile Ile Thr His Pro Asn Phe Asn Gly Asn Thr
65 70 75 80
Leu Asp Asn Asp Ile Met Leu Ile Lys Leu Ser Ser Pro Ala Thr Leu
85 90 95
Asn Ser Arg Val Ala Thr Val Ser Leu Pro Arg Ser Cys Ala Ala Ala
100 105 110
Gly Thr Glu Cys Leu Ile Ser Gly Trp Gly Asn Thr Lys Ser Ser Gly
115 120 125
Ser Ser Tyr Pro Ser Leu Leu Gln Cys Leu Lys Ala Pro Val Leu Ser
130 135 140
Asp Ser Ser Cys Lys Ser Ser Tyr Pro Gly Gln Ile Thr Gly Asn Met
145 150 155 160
Ile Cys Val Gly Phe Leu Glu Gly Gly Asn Asp Ala Cys Gln Gly Asp
165 170 175
Ser Gly Gly Pro Val Val Cys Asn Gly Gln Leu Gln Gly Ile Val Ser
180 185 190
Ser Gly Tyr Gly Cys Ala Gln Lys Asn Lys Pro Gly Val Tyr Thr Lys
195 200 205
Val Cys Asn Tyr Val Asn Trp Ile Gln Gln Thr Ile Ala Ala Asn
210 215 220
<210> 28
<211> 223
<212> PRT
<213> 人工序列
<220>
<223> 猪胰蛋白酶变体28号
<400> 28
Ile Val Gly Gly Tyr Thr Cys Ala Ala Asn Ser Ile Pro Tyr Gln Val
1 5 10 15
Ser Leu Asn Ser Gly Ser His Val Cys Gly Gly Ser Leu Ile Asn Ser
20 25 30
Gln Trp Val Val Ser Ala Ala His Cys Tyr Lys Ser Arg Ile Gln Val
35 40 45
Arg Leu Gly Glu His Asn Ile Asp Val Leu Glu Gly Asn Glu Gln Phe
50 55 60
Ile Asn Ala Ala Lys Ile Ile Thr His Pro Asn Phe Asn Gly Asn Thr
65 70 75 80
Leu Asp Asn Asp Ile Met Leu Ile Lys Leu Ser Ser Pro Ala Thr Leu
85 90 95
Asn Ser Arg Val Ala Thr Val Ser Leu Pro Arg Ser Cys Ala Ala Ala
100 105 110
Gly Thr Glu Cys Leu Ile Ser Gly Trp Gly Asn Thr Lys Ser Ser Gly
115 120 125
Ser Ser Tyr Pro Ser Leu Leu Gln Cys Leu Lys Ala Pro Val Leu Ser
130 135 140
Asp Ser Ser Cys Lys Ser Ser Tyr Pro Gly Gln Ile Thr Gly Asn Met
145 150 155 160
Ile Cys Val Gly Phe Leu Glu Gly Gly Lys Asp Ala Cys Gln Gly Asp
165 170 175
Ser Gly Gly Pro Val Val Cys Asn Gly Gln Leu Gln Gly Ile Val Ser
180 185 190
Ser Gly Tyr Gly Cys Ala Gln Lys Asn Lys Pro Gly Val Tyr Thr Lys
195 200 205
Val Cys Asn Tyr Val Asn Trp Ile Gln Gln Thr Ile Ala Ala Asn
210 215 220
<210> 29
<211> 223
<212> PRT
<213> 人工序列
<220>
<223> 猪胰蛋白酶变体29号
<400> 29
Ile Val Gly Gly Tyr Thr Cys Ala Ala Asn Ser Ile Pro Tyr Gln Val
1 5 10 15
Ser Leu Asn Ser Gly Ser His Leu Cys Gly Gly Ser Leu Ile Asn Ser
20 25 30
Gln Trp Val Val Ser Ala Ala His Cys Tyr Lys Ser Arg Ile Gln Val
35 40 45
Arg Leu Gly Glu His Asn Ile Asp Val Leu Glu Gly Asn Glu Gln Phe
50 55 60
Ile Asn Ala Ala Lys Ile Ile Thr His Pro Asn Phe Asn Gly Asn Thr
65 70 75 80
Leu Asp Asn Asp Ile Met Leu Ile Lys Leu Ser Ser Pro Ala Thr Leu
85 90 95
Asn Ser Arg Val Ala Thr Val Ser Leu Pro Arg Ser Cys Ala Ala Ala
100 105 110
Gly Thr Glu Cys Leu Ile Ser Gly Trp Gly Asn Thr Lys Ser Ser Gly
115 120 125
Ser Ser Tyr Pro Ser Leu Leu Gln Cys Leu Lys Ala Pro Val Leu Ser
130 135 140
Asp Ser Ser Cys Lys Ser Ser Tyr Pro Gly Gln Ile Thr Gly Asn Met
145 150 155 160
Ile Cys Val Gly Phe Leu Glu Gly Gly Lys Asp Ala Cys Gln Gly Asp
165 170 175
Ser Gly Gly Pro Val Val Cys Asn Gly Gln Leu Gln Gly Ile Val Ser
180 185 190
Ser Gly Tyr Gly Cys Ala Gln Lys Asn Lys Pro Gly Val Tyr Thr Lys
195 200 205
Val Cys Asn Tyr Val Asn Trp Ile Gln Gln Thr Ile Ala Ala Asn
210 215 220
<210> 30
<211> 223
<212> PRT
<213> 人工序列
<220>
<223> 猪胰蛋白酶变体30号
<400> 30
Ile Val Gly Gly Tyr Thr Cys Ala Ala Asn Ser Ile Pro Tyr Gln Val
1 5 10 15
Ser Leu Asn Ser Gly Ser His Ser Cys Gly Gly Ser Leu Ile Asn Ser
20 25 30
Gln Trp Val Val Ser Ala Ala His Cys Tyr Lys Ser Arg Ile Gln Val
35 40 45
Arg Leu Gly Glu His Asn Ile Asp Val Leu Glu Gly Asn Glu Gln Phe
50 55 60
Ile Asn Ala Ala Lys Ile Ile Thr His Pro Asn Phe Asn Gly Asn Thr
65 70 75 80
Leu Asp Asn Asp Ile Met Leu Ile Lys Leu Ser Ser Pro Ala Thr Leu
85 90 95
Asn Ser Arg Val Ala Thr Val Ser Leu Pro Arg Ser Cys Ala Ala Ala
100 105 110
Gly Thr Glu Cys Leu Ile Ser Gly Trp Gly Asn Thr Lys Ser Ser Gly
115 120 125
Ser Ser Tyr Pro Ser Leu Leu Gln Cys Leu Lys Ala Pro Val Leu Ser
130 135 140
Asp Ser Ser Cys Lys Ser Ser Tyr Pro Gly Gln Ile Thr Gly Asn Met
145 150 155 160
Ile Cys Val Gly Phe Leu Glu Gly Gly Lys Asp Ala Cys Gln Gly Asp
165 170 175
Ser Gly Gly Pro Val Val Cys Asn Gly Gln Leu Gln Gly Ile Val Ser
180 185 190
Ser Gly Tyr Gly Cys Ala Gln Lys Asn Lys Pro Gly Val Tyr Thr Lys
195 200 205
Val Cys Asn Tyr Val Asn Trp Ile Gln Gln Thr Ile Ala Ala Asn
210 215 220
<210> 31
<211> 223
<212> PRT
<213> 人工序列
<220>
<223> 猪胰蛋白酶变体31号
<400> 31
Ile Val Gly Gly Tyr Thr Cys Ala Ala Asn Ser Ile Pro Tyr Gln Val
1 5 10 15
Ser Leu Asn Ser Gly Ser His Gln Cys Gly Gly Ser Leu Ile Asn Ser
20 25 30
Gln Trp Val Val Ser Ala Ala His Cys Tyr Lys Ser Arg Ile Gln Val
35 40 45
Arg Leu Gly Glu His Asn Ile Asp Val Leu Glu Gly Asn Glu Gln Phe
50 55 60
Ile Asn Ala Ala Lys Ile Ile Thr His Pro Asn Phe Asn Gly Asn Thr
65 70 75 80
Leu Asp Asn Asp Ile Met Leu Ile Lys Leu Ser Ser Pro Ala Thr Leu
85 90 95
Asn Ser Arg Val Ala Thr Val Ser Leu Pro Arg Ser Cys Ala Ala Ala
100 105 110
Gly Thr Glu Cys Leu Ile Ser Gly Trp Gly Asn Thr Lys Ser Ser Gly
115 120 125
Ser Ser Tyr Pro Ser Leu Leu Gln Cys Leu Lys Ala Pro Val Leu Ser
130 135 140
Asp Ser Ser Cys Lys Ser Ser Tyr Pro Gly Gln Ile Thr Gly Asn Met
145 150 155 160
Ile Cys Val Gly Phe Leu Glu Gly Gly Lys Asp Ala Cys Gln Gly Asp
165 170 175
Ser Gly Gly Pro Val Val Cys Asn Gly Gln Leu Gln Gly Ile Val Ser
180 185 190
Ser Gly Tyr Gly Cys Ala Gln Lys Asn Lys Pro Gly Val Tyr Thr Lys
195 200 205
Val Cys Asn Tyr Val Asn Trp Ile Gln Gln Thr Ile Ala Ala Asn
210 215 220
<210> 32
<211> 223
<212> PRT
<213> 人工序列
<220>
<223> 猪胰蛋白酶变体32号
<400> 32
Ile Val Gly Gly Tyr Thr Cys Ala Ala Asn Ser Ile Pro Tyr Gln Val
1 5 10 15
Ser Leu Asn Ser Gly Ser His Arg Cys Gly Gly Ser Leu Ile Asn Ser
20 25 30
Gln Trp Val Val Ser Ala Ala His Cys Tyr Lys Ser Arg Ile Gln Val
35 40 45
Arg Leu Gly Glu His Asn Ile Asp Val Leu Glu Gly Asn Glu Gln Phe
50 55 60
Ile Asn Ala Ala Lys Ile Ile Thr His Pro Asn Phe Asn Gly Asn Thr
65 70 75 80
Leu Asp Asn Asp Ile Met Leu Ile Lys Leu Ser Ser Pro Ala Thr Leu
85 90 95
Asn Ser Arg Val Ala Thr Val Ser Leu Pro Arg Ser Cys Ala Ala Ala
100 105 110
Gly Thr Glu Cys Leu Ile Ser Gly Trp Gly Asn Thr Lys Ser Ser Gly
115 120 125
Ser Ser Tyr Pro Ser Leu Leu Gln Cys Leu Lys Ala Pro Val Leu Ser
130 135 140
Asp Ser Ser Cys Lys Ser Ser Tyr Pro Gly Gln Ile Thr Gly Asn Met
145 150 155 160
Ile Cys Val Gly Phe Leu Glu Gly Gly Lys Asp Ala Cys Gln Gly Asp
165 170 175
Ser Gly Gly Pro Val Val Cys Asn Gly Gln Leu Gln Gly Ile Val Ser
180 185 190
Ser Gly Tyr Gly Cys Ala Gln Lys Asn Lys Pro Gly Val Tyr Thr Lys
195 200 205
Val Cys Asn Tyr Val Asn Trp Ile Gln Gln Thr Ile Ala Ala Asn
210 215 220
<210> 33
<211> 223
<212> PRT
<213> 人工序列
<220>
<223> 猪胰蛋白酶变体33号
<400> 33
Ile Val Gly Gly Tyr Thr Cys Ala Ala Asn Ser Ile Pro Tyr Gln Val
1 5 10 15
Ser Leu Asn Ser Gly Ser His Arg Cys Gly Gly Ser Leu Ile Asn Ser
20 25 30
Gln Trp Val Val Ser Ala Ala His Cys Tyr Lys Ser Arg Ile Gln Val
35 40 45
Arg Leu Gly Glu His Asn Ile Asp Val Leu Glu Gly Asn Glu Gln Phe
50 55 60
Ile Asn Ala Ala Lys Ile Ile Thr His Pro Asn Phe Asn Gly Asn Thr
65 70 75 80
Leu Asp Asn Asp Ile Met Leu Ile Lys Leu Ser Ser Pro Ala Thr Leu
85 90 95
Asn Ser Arg Val Ala Thr Val Ser Leu Pro Thr Ser Cys Ala Ala Ala
100 105 110
Gly Thr Glu Cys Leu Ile Ser Gly Trp Gly Asn Thr Lys Ser Ser Gly
115 120 125
Ser Ser Tyr Pro Ser Leu Leu Gln Cys Leu Lys Ala Pro Val Leu Ser
130 135 140
Asp Ser Ser Cys Lys Ser Ser Tyr Pro Gly Gln Ile Thr Gly Asn Met
145 150 155 160
Ile Cys Val Gly Phe Leu Glu Gly Gly Lys Asp Ala Cys Gln Gly Asp
165 170 175
Ser Gly Gly Pro Val Val Cys Asn Gly Gln Leu Gln Gly Ile Val Ser
180 185 190
Ser Gly Tyr Gly Cys Ala Gln Lys Asn Lys Pro Gly Val Tyr Thr Lys
195 200 205
Val Cys Asn Tyr Val Asn Trp Ile Gln Gln Thr Ile Ala Ala Asn
210 215 220
<210> 34
<211> 223
<212> PRT
<213> 人工序列
<220>
<223> 猪胰蛋白酶变体34号
<400> 34
Ile Val Gly Gly Tyr Thr Cys Ala Ala Asn Ser Ile Pro Tyr Gln Val
1 5 10 15
Ser Leu Asn Ser Gly Ser His Arg Cys Gly Gly Ser Leu Ile Asn Ser
20 25 30
Gln Trp Val Val Ser Ala Ala His Cys Tyr Lys Ser Arg Ile Gln Val
35 40 45
Arg Leu Gly Glu His Asn Ile Asp Val Leu Glu Gly Asn Glu Gln Phe
50 55 60
Ile Asn Ala Ala Lys Ile Ile Thr His Pro Asn Phe Asn Gly Asn Thr
65 70 75 80
Leu Asp Asn Asp Ile Met Leu Ile Lys Leu Ser Ser Pro Ala Thr Leu
85 90 95
Asn Ser Arg Val Ala Thr Val Ser Leu Pro Arg Ser Cys Ala Ala Ala
100 105 110
Gly Thr Glu Cys Leu Ile Ser Gly Trp Gly Asn Thr Lys Ser Ser Gly
115 120 125
Ser Ser Ala Pro Ser Leu Leu Gln Cys Leu Lys Ala Pro Val Leu Ser
130 135 140
Asp Ser Ser Cys Lys Ser Ser Tyr Pro Gly Gln Ile Thr Gly Asn Met
145 150 155 160
Ile Cys Val Gly Phe Leu Glu Gly Gly Lys Asp Ala Cys Gln Gly Asp
165 170 175
Ser Gly Gly Pro Val Val Cys Asn Gly Gln Leu Gln Gly Ile Val Ser
180 185 190
Ser Gly Tyr Gly Cys Ala Gln Lys Asn Lys Pro Gly Val Tyr Thr Lys
195 200 205
Val Cys Asn Tyr Val Asn Trp Ile Gln Gln Thr Ile Ala Ala Asn
210 215 220
<210> 35
<211> 223
<212> PRT
<213> 人工序列
<220>
<223> 猪胰蛋白酶变体35号
<400> 35
Ile Val Gly Gly Tyr Thr Cys Ala Ala Asn Ser Ile Pro Tyr Gln Val
1 5 10 15
Ser Leu Asn Ser Gly Ser His Arg Cys Gly Gly Ser Leu Ile Asn Ser
20 25 30
Gln Trp Val Val Ser Ala Ala His Cys Tyr Lys Ser Arg Ile Gln Val
35 40 45
Arg Leu Gly Glu His Asn Ile Asp Val Leu Glu Gly Asn Glu Gln Phe
50 55 60
Ile Asn Ala Ala Lys Ile Ile Thr His Pro Asn Phe Asn Gly Asn Thr
65 70 75 80
Leu Asp Asn Asp Ile Met Leu Ile Lys Leu Ser Ser Pro Ala Thr Leu
85 90 95
Asn Ser Arg Val Ala Thr Val Ser Leu Pro Arg Ser Cys Ala Ala Ala
100 105 110
Gly Thr Glu Cys Leu Ile Ser Gly Trp Gly Asn Thr Lys Ser Ser Gly
115 120 125
Ser Ser Leu Pro Ser Leu Leu Gln Cys Leu Lys Ala Pro Val Leu Ser
130 135 140
Asp Ser Ser Cys Lys Ser Ser Tyr Pro Gly Gln Ile Thr Gly Asn Met
145 150 155 160
Ile Cys Val Gly Phe Leu Glu Gly Gly Lys Asp Ala Cys Gln Gly Asp
165 170 175
Ser Gly Gly Pro Val Val Cys Asn Gly Gln Leu Gln Gly Ile Val Ser
180 185 190
Ser Gly Tyr Gly Cys Ala Gln Lys Asn Lys Pro Gly Val Tyr Thr Lys
195 200 205
Val Cys Asn Tyr Val Asn Trp Ile Gln Gln Thr Ile Ala Ala Asn
210 215 220
<210> 36
<211> 223
<212> PRT
<213> 人工序列
<220>
<223> 猪胰蛋白酶变体36号
<400> 36
Ile Val Gly Gly Tyr Thr Cys Ala Ala Asn Ser Ile Pro Tyr Gln Val
1 5 10 15
Ser Leu Asn Ser Gly Ser His Arg Cys Gly Gly Ser Leu Ile Asn Ser
20 25 30
Gln Trp Val Val Ser Ala Ala His Cys Tyr Lys Ser Arg Ile Gln Val
35 40 45
Arg Leu Gly Glu His Asn Ile Asp Val Leu Glu Gly Asn Glu Gln Phe
50 55 60
Ile Asn Ala Ala Lys Ile Ile Thr His Pro Asn Phe Asn Gly Asn Thr
65 70 75 80
Leu Asp Asn Asp Ile Met Leu Ile Lys Leu Ser Ser Pro Ala Thr Leu
85 90 95
Asn Ser Arg Val Ala Thr Val Ser Leu Pro Arg Ser Cys Ala Ala Ala
100 105 110
Gly Thr Glu Cys Leu Ile Ser Gly Trp Gly Asn Thr Lys Ser Ser Gly
115 120 125
Ser Ser Met Pro Ser Leu Leu Gln Cys Leu Lys Ala Pro Val Leu Ser
130 135 140
Asp Ser Ser Cys Lys Ser Ser Tyr Pro Gly Gln Ile Thr Gly Asn Met
145 150 155 160
Ile Cys Val Gly Phe Leu Glu Gly Gly Lys Asp Ala Cys Gln Gly Asp
165 170 175
Ser Gly Gly Pro Val Val Cys Asn Gly Gln Leu Gln Gly Ile Val Ser
180 185 190
Ser Gly Tyr Gly Cys Ala Gln Lys Asn Lys Pro Gly Val Tyr Thr Lys
195 200 205
Val Cys Asn Tyr Val Asn Trp Ile Gln Gln Thr Ile Ala Ala Asn
210 215 220
<210> 37
<211> 223
<212> PRT
<213> 人工序列
<220>
<223> 猪胰蛋白酶变体37号
<400> 37
Ile Val Gly Gly Tyr Thr Cys Ala Ala Asn Ser Ile Pro Tyr Gln Val
1 5 10 15
Ser Leu Asn Ser Gly Ser His Arg Cys Gly Gly Ser Leu Ile Asn Ser
20 25 30
Gln Trp Val Val Ser Ala Ala His Cys Tyr Lys Ser Arg Ile Gln Val
35 40 45
Arg Leu Gly Glu His Asn Ile Asp Val Leu Glu Gly Asn Glu Gln Phe
50 55 60
Ile Asn Ala Ala Lys Ile Ile Thr His Pro Asn Phe Asn Gly Asn Thr
65 70 75 80
Leu Asp Asn Asp Ile Met Leu Ile Lys Leu Ser Ser Pro Ala Thr Leu
85 90 95
Asn Ser Arg Val Ala Thr Val Ser Leu Pro Arg Ser Cys Ala Ala Ala
100 105 110
Gly Thr Glu Cys Leu Ile Ser Gly Trp Gly Asn Thr Lys Ser Ser Gly
115 120 125
Ser Ser Thr Pro Ser Leu Leu Gln Cys Leu Lys Ala Pro Val Leu Ser
130 135 140
Asp Ser Ser Cys Lys Ser Ser Tyr Pro Gly Gln Ile Thr Gly Asn Met
145 150 155 160
Ile Cys Val Gly Phe Leu Glu Gly Gly Lys Asp Ala Cys Gln Gly Asp
165 170 175
Ser Gly Gly Pro Val Val Cys Asn Gly Gln Leu Gln Gly Ile Val Ser
180 185 190
Ser Gly Tyr Gly Cys Ala Gln Lys Asn Lys Pro Gly Val Tyr Thr Lys
195 200 205
Val Cys Asn Tyr Val Asn Trp Ile Gln Gln Thr Ile Ala Ala Asn
210 215 220
<210> 38
<211> 223
<212> PRT
<213> 人工序列
<220>
<223> 猪胰蛋白酶变体38号
<400> 38
Ile Val Gly Gly Tyr Thr Cys Ala Ala Asn Ser Ile Pro Tyr Gln Val
1 5 10 15
Ser Leu Asn Ser Gly Ser His Arg Cys Gly Gly Ser Leu Ile Asn Ser
20 25 30
Gln Trp Val Val Ser Ala Ala His Cys Tyr Lys Ser Arg Ile Gln Val
35 40 45
Arg Leu Gly Glu His Asn Ile Asp Val Leu Glu Gly Asn Glu Gln Phe
50 55 60
Ile Asn Ala Ala Lys Ile Ile Thr His Pro Asn Phe Asn Gly Asn Thr
65 70 75 80
Leu Asp Asn Asp Ile Met Leu Ile Lys Leu Ser Ser Pro Ala Thr Leu
85 90 95
Asn Ser Arg Val Ala Thr Val Ser Leu Pro Arg Ser Cys Ala Ala Ala
100 105 110
Gly Thr Glu Cys Leu Ile Ser Gly Trp Gly Asn Thr Lys Ser Ser Gly
115 120 125
Ser Ser Cys Pro Ser Leu Leu Gln Cys Leu Lys Ala Pro Val Leu Ser
130 135 140
Asp Ser Ser Cys Lys Ser Ser Tyr Pro Gly Gln Ile Thr Gly Asn Met
145 150 155 160
Ile Cys Val Gly Phe Leu Glu Gly Gly Lys Asp Ala Cys Gln Gly Asp
165 170 175
Ser Gly Gly Pro Val Val Cys Asn Gly Gln Leu Gln Gly Ile Val Ser
180 185 190
Ser Gly Tyr Gly Cys Ala Gln Lys Asn Lys Pro Gly Val Tyr Thr Lys
195 200 205
Val Cys Asn Tyr Val Asn Trp Ile Gln Gln Thr Ile Ala Ala Asn
210 215 220
<210> 39
<211> 223
<212> PRT
<213> 人工序列
<220>
<223> 猪胰蛋白酶变体39号
<400> 39
Ile Val Gly Gly Tyr Thr Cys Ala Ala Asn Ser Ile Pro Tyr Gln Val
1 5 10 15
Ser Leu Asn Ser Gly Ser His Arg Cys Gly Gly Ser Leu Ile Asn Ser
20 25 30
Gln Trp Val Val Ser Ala Ala His Cys Tyr Lys Ser Arg Ile Gln Val
35 40 45
Arg Leu Gly Glu His Asn Ile Asp Val Leu Glu Gly Asn Glu Gln Phe
50 55 60
Ile Asn Ala Ala Lys Ile Ile Thr His Pro Asn Phe Asn Gly Asn Thr
65 70 75 80
Leu Asp Asn Asp Ile Met Leu Ile Lys Leu Ser Ser Pro Ala Thr Leu
85 90 95
Asn Ser Arg Val Ala Thr Val Ser Leu Pro Arg Ser Cys Ala Ala Ala
100 105 110
Gly Thr Glu Cys Leu Ile Ser Gly Trp Gly Asn Thr Lys Ser Ser Gly
115 120 125
Ser Ser Asn Pro Ser Leu Leu Gln Cys Leu Lys Ala Pro Val Leu Ser
130 135 140
Asp Ser Ser Cys Lys Ser Ser Tyr Pro Gly Gln Ile Thr Gly Asn Met
145 150 155 160
Ile Cys Val Gly Phe Leu Glu Gly Gly Lys Asp Ala Cys Gln Gly Asp
165 170 175
Ser Gly Gly Pro Val Val Cys Asn Gly Gln Leu Gln Gly Ile Val Ser
180 185 190
Ser Gly Tyr Gly Cys Ala Gln Lys Asn Lys Pro Gly Val Tyr Thr Lys
195 200 205
Val Cys Asn Tyr Val Asn Trp Ile Gln Gln Thr Ile Ala Ala Asn
210 215 220
<210> 40
<211> 223
<212> PRT
<213> 人工序列
<220>
<223> 猪胰蛋白酶变体40号
<400> 40
Ile Val Gly Gly Tyr Thr Cys Ala Ala Asn Ser Ile Pro Tyr Gln Val
1 5 10 15
Ser Leu Asn Ser Gly Ser His Arg Cys Gly Gly Ser Leu Ile Asn Ser
20 25 30
Gln Trp Val Val Ser Ala Ala His Cys Tyr Lys Ser Arg Ile Gln Val
35 40 45
Arg Leu Gly Glu His Asn Ile Asp Val Leu Glu Gly Asn Glu Gln Phe
50 55 60
Ile Asn Ala Ala Lys Ile Ile Thr His Pro Asn Phe Asn Gly Asn Thr
65 70 75 80
Leu Asp Asn Asp Ile Met Leu Ile Lys Leu Ser Ser Pro Ala Thr Leu
85 90 95
Asn Ser Arg Val Ala Thr Val Ser Leu Pro Arg Ser Cys Ala Ala Ala
100 105 110
Gly Thr Glu Cys Leu Ile Ser Gly Trp Gly Asn Thr Lys Ser Ser Gly
115 120 125
Ser Ser Glu Pro Ser Leu Leu Gln Cys Leu Lys Ala Pro Val Leu Ser
130 135 140
Asp Ser Ser Cys Lys Ser Ser Tyr Pro Gly Gln Ile Thr Gly Asn Met
145 150 155 160
Ile Cys Val Gly Phe Leu Glu Gly Gly Lys Asp Ala Cys Gln Gly Asp
165 170 175
Ser Gly Gly Pro Val Val Cys Asn Gly Gln Leu Gln Gly Ile Val Ser
180 185 190
Ser Gly Tyr Gly Cys Ala Gln Lys Asn Lys Pro Gly Val Tyr Thr Lys
195 200 205
Val Cys Asn Tyr Val Asn Trp Ile Gln Gln Thr Ile Ala Ala Asn
210 215 220
<210> 41
<211> 223
<212> PRT
<213> 人工序列
<220>
<223> 猪胰蛋白酶变体41号
<400> 41
Ile Val Gly Gly Tyr Thr Cys Ala Ala Asn Ser Ile Pro Tyr Gln Val
1 5 10 15
Ser Leu Asn Ser Gly Ser His Arg Cys Gly Gly Ser Leu Ile Asn Ser
20 25 30
Gln Trp Val Val Ser Ala Ala His Cys Tyr Lys Ser Arg Ile Gln Val
35 40 45
Arg Leu Gly Glu His Asn Ile Asp Val Leu Glu Gly Asn Glu Gln Phe
50 55 60
Ile Asn Ala Ala Lys Ile Ile Thr His Pro Asn Phe Asn Gly Asn Thr
65 70 75 80
Leu Asp Asn Asp Ile Met Leu Ile Lys Leu Ser Ser Pro Ala Thr Leu
85 90 95
Asn Ser Arg Val Ala Thr Val Ser Leu Pro Arg Ser Cys Ala Ala Ala
100 105 110
Gly Thr Glu Cys Leu Ile Ser Gly Trp Gly Asn Thr Lys Ser Ser Gly
115 120 125
Ser Ser His Pro Ser Leu Leu Gln Cys Leu Lys Ala Pro Val Leu Ser
130 135 140
Asp Ser Ser Cys Lys Ser Ser Tyr Pro Gly Gln Ile Thr Gly Asn Met
145 150 155 160
Ile Cys Val Gly Phe Leu Glu Gly Gly Lys Asp Ala Cys Gln Gly Asp
165 170 175
Ser Gly Gly Pro Val Val Cys Asn Gly Gln Leu Gln Gly Ile Val Ser
180 185 190
Ser Gly Tyr Gly Cys Ala Gln Lys Asn Lys Pro Gly Val Tyr Thr Lys
195 200 205
Val Cys Asn Tyr Val Asn Trp Ile Gln Gln Thr Ile Ala Ala Asn
210 215 220
<210> 42
<211> 223
<212> PRT
<213> 人工序列
<220>
<223> 猪胰蛋白酶变体42号
<400> 42
Ile Val Gly Gly Tyr Thr Cys Ala Ala Asn Ser Ile Pro Tyr Gln Val
1 5 10 15
Ser Leu Asn Ser Gly Ser His Arg Cys Gly Gly Ser Leu Ile Asn Ser
20 25 30
Gln Trp Val Val Ser Ala Ala His Cys Tyr Lys Ser Arg Ile Gln Val
35 40 45
Arg Leu Gly Glu His Asn Ile Asp Val Leu Glu Gly Asn Glu Gln Phe
50 55 60
Ile Asn Ala Ala Lys Ile Ile Thr His Pro Asn Phe Asn Gly Asn Thr
65 70 75 80
Leu Asp Asn Asp Ile Met Leu Ile Lys Leu Ser Ser Pro Ala Thr Leu
85 90 95
Asn Ser Arg Val Ala Thr Val Ser Leu Pro Arg Ser Cys Ala Ala Ala
100 105 110
Gly Thr Glu Cys Leu Ile Ser Gly Trp Gly Asn Thr Lys Ser Ser Gly
115 120 125
Ser Ser Tyr Pro Ser Leu Leu Gln Cys Leu Lys Ala Pro Val Leu Ser
130 135 140
Asp Ser Ser Cys Lys Ser Ser Tyr Pro Gly Gln Ile Thr Gly Asn Met
145 150 155 160
Ile Cys Val Gly Phe Leu Glu Gly Gly Lys Asp Ala Cys Gln Gly Asp
165 170 175
Ser Gly Gly Pro Val Val Cys Asn Gly Gln Leu Gln Gly Ile Val Ser
180 185 190
Ser Gly Tyr Gly Cys Ala Gln Lys Asn Lys Pro Gly Val Tyr Thr Lys
195 200 205
Val Cys Asn Tyr Val Asn Trp Ile Gln Gln Thr Ile Ala Ala Asn
210 215 220
<210> 43
<211> 223
<212> PRT
<213> 人工序列
<220>
<223> 猪胰蛋白酶变体43号
<400> 43
Ile Val Gly Gly Tyr Thr Cys Ala Ala Asn Ser Ile Pro Tyr Gln Val
1 5 10 15
Ser Leu Asn Ser Gly Ser His Arg Cys Gly Gly Ser Leu Ile Asn Ser
20 25 30
Gln Trp Val Val Ser Ala Ala His Cys Tyr Lys Ser Arg Ile Gln Val
35 40 45
Arg Leu Gly Glu His Asn Ile Asp Val Leu Glu Gly Asn Glu Gln Phe
50 55 60
Ile Asn Ala Ala Lys Ile Ile Thr His Pro Asn Phe Asn Gly Asn Thr
65 70 75 80
Leu Asp Asn Asp Ile Met Leu Ile Lys Leu Ser Ser Pro Ala Thr Leu
85 90 95
Asn Ser Arg Val Ala Thr Val Ser Leu Pro Arg Ser Cys Ala Ala Ala
100 105 110
Gly Thr Glu Cys Leu Ile Ser Gly Trp Gly Asn Thr Lys Ser Ser Gly
115 120 125
Ser Ser Tyr Pro Ser Leu Leu Gln Cys Leu Lys Ala Pro Val Leu Ser
130 135 140
Asp Ser Ser Cys Lys Ser Ser Tyr Pro Gly Gln Ile Thr Gly Asn Met
145 150 155 160
Ile Cys Val Gly Phe Leu Glu Gly Gly Lys Asp Thr Cys Gln Gly Asp
165 170 175
Ser Gly Gly Pro Val Val Cys Asn Gly Gln Leu Gln Gly Ile Val Ser
180 185 190
Ser Gly Tyr Gly Cys Ala Gln Lys Asn Lys Pro Gly Val Tyr Thr Lys
195 200 205
Val Cys Asn Tyr Val Asn Trp Ile Gln Gln Thr Ile Ala Ala Asn
210 215 220
<210> 44
<211> 223
<212> PRT
<213> 人工序列
<220>
<223> 猪胰蛋白酶变体44号
<400> 44
Ile Val Gly Gly Tyr Thr Cys Ala Ala Asn Ser Ile Pro Tyr Gln Val
1 5 10 15
Ser Leu Asn Ser Gly Ser His Arg Cys Gly Gly Ser Leu Ile Asn Ser
20 25 30
Gln Trp Val Val Ser Ala Ala His Cys Tyr Lys Ser Arg Ile Gln Val
35 40 45
Arg Leu Gly Glu His Asn Ile Asp Val Leu Glu Gly Asn Glu Gln Phe
50 55 60
Ile Asn Ala Ala Lys Ile Ile Thr His Pro Asn Phe Asn Gly Asn Thr
65 70 75 80
Leu Asp Asn Asp Ile Met Leu Ile Lys Leu Ser Ser Pro Ala Thr Leu
85 90 95
Asn Ser Arg Val Ala Thr Val Ser Leu Pro Arg Ser Cys Ala Ala Ala
100 105 110
Gly Thr Glu Cys Leu Ile Ser Gly Trp Gly Asn Thr Lys Ser Ser Gly
115 120 125
Ser Ser Tyr Pro Ser Leu Leu Gln Cys Leu Lys Ala Pro Val Leu Ser
130 135 140
Asp Ser Ser Cys Lys Ser Ser Tyr Pro Gly Gln Ile Thr Gly Asn Met
145 150 155 160
Ile Cys Val Gly Phe Leu Glu Gly Gly Lys Asp Ser Cys Gln Gly Asp
165 170 175
Ser Gly Gly Pro Val Val Cys Asn Gly Gln Leu Gln Gly Ile Val Ser
180 185 190
Ser Gly Tyr Gly Cys Ala Gln Lys Asn Lys Pro Gly Val Tyr Thr Lys
195 200 205
Val Cys Asn Tyr Val Asn Trp Ile Gln Gln Thr Ile Ala Ala Asn
210 215 220
<210> 45
<211> 223
<212> PRT
<213> 人工序列
<220>
<223> 猪胰蛋白酶变体45号
<400> 45
Ile Val Gly Gly Tyr Thr Cys Ala Ala Asn Ser Ile Pro Tyr Gln Val
1 5 10 15
Ser Leu Asn Ser Gly Ser His Arg Cys Gly Gly Ser Leu Ile Asn Ser
20 25 30
Gln Trp Val Val Ser Ala Ala His Cys Tyr Lys Ser Arg Ile Gln Val
35 40 45
Arg Leu Gly Glu His Asn Ile Asp Val Leu Glu Gly Asn Glu Gln Phe
50 55 60
Ile Asn Ala Ala Lys Ile Ile Thr His Pro Asn Phe Asn Gly Asn Thr
65 70 75 80
Leu Asp Asn Asp Ile Met Leu Ile Lys Leu Ser Ser Pro Ala Thr Leu
85 90 95
Asn Ser Arg Val Ala Thr Val Ser Leu Pro Arg Ser Cys Ala Ala Ala
100 105 110
Gly Thr Glu Cys Leu Ile Ser Gly Trp Gly Asn Thr Lys Ser Ser Gly
115 120 125
Ser Ser Tyr Pro Ser Leu Leu Gln Cys Leu Lys Ala Pro Val Leu Ser
130 135 140
Asp Ser Ser Cys Lys Ser Ser Tyr Pro Gly Gln Ile Thr Gly Asn Met
145 150 155 160
Ile Cys Val Gly Phe Leu Glu Gly Gly Lys Asp Cys Cys Gln Gly Asp
165 170 175
Ser Gly Gly Pro Val Val Cys Asn Gly Gln Leu Gln Gly Ile Val Ser
180 185 190
Ser Gly Tyr Gly Cys Ala Gln Lys Asn Lys Pro Gly Val Tyr Thr Lys
195 200 205
Val Cys Asn Tyr Val Asn Trp Ile Gln Gln Thr Ile Ala Ala Asn
210 215 220
<210> 46
<211> 223
<212> PRT
<213> 人工序列
<220>
<223> 猪胰蛋白酶变体46号
<400> 46
Ile Val Gly Gly Tyr Thr Cys Ala Ala Asn Ser Ile Pro Tyr Gln Val
1 5 10 15
Ser Leu Asn Ser Gly Ser His Ala Cys Gly Gly Ser Leu Ile Asn Ser
20 25 30
Gln Trp Val Val Ser Ala Ala His Cys Tyr Lys Ser Arg Ile Gln Val
35 40 45
Arg Leu Gly Glu His Asn Ile Asp Val Leu Glu Gly Asn Glu Gln Phe
50 55 60
Ile Asn Ala Ala Lys Ile Ile Thr His Pro Asn Phe Asn Gly Asn Thr
65 70 75 80
Leu Asp Asn Asp Ile Met Leu Ile Lys Leu Ser Ser Pro Ala Thr Leu
85 90 95
Asn Ser Arg Val Ala Thr Val Ser Leu Pro Arg Ser Cys Ala Ala Ala
100 105 110
Gly Thr Glu Cys Leu Ile Ser Gly Trp Gly Asn Thr Lys Ser Ser Gly
115 120 125
Ser Ser Tyr Pro Ser Leu Leu Gln Cys Leu Lys Ala Pro Val Leu Ser
130 135 140
Asp Ser Ser Cys Lys Ser Ser Tyr Pro Gly Gln Ile Thr Gly Asn Met
145 150 155 160
Ile Cys Val Gly Phe Leu Glu Gly Gly Lys Asp Ala Cys Gln Gly Asp
165 170 175
Ser Gly Gly Pro Val Val Cys Asn Gly Gln Leu Gln Gly Ile Val Ser
180 185 190
Ser Gly Tyr Gly Cys Ala Gln Lys Asn Lys Pro Gly Val Tyr Thr Lys
195 200 205
Val Cys Asn Tyr Val Asn Trp Ile Gln Gln Thr Ile Ala Ala Asn
210 215 220
<210> 47
<211> 223
<212> PRT
<213> 人工序列
<220>
<223> 猪胰蛋白酶变体47号
<400> 47
Ile Val Gly Gly Tyr Thr Cys Ala Ala Asn Ser Ile Pro Tyr Gln Val
1 5 10 15
Ser Leu Asn Ser Gly Ser His Ile Cys Gly Gly Ser Leu Ile Asn Ser
20 25 30
Gln Trp Val Val Ser Ala Ala His Cys Tyr Lys Ser Arg Ile Gln Val
35 40 45
Arg Leu Gly Glu His Asn Ile Asp Val Leu Glu Gly Asn Glu Gln Phe
50 55 60
Ile Asn Ala Ala Lys Ile Ile Thr His Pro Asn Phe Asn Gly Asn Thr
65 70 75 80
Leu Asp Asn Asp Ile Met Leu Ile Lys Leu Ser Ser Pro Ala Thr Leu
85 90 95
Asn Ser Arg Val Ala Thr Val Ser Leu Pro Arg Ser Cys Ala Ala Ala
100 105 110
Gly Thr Glu Cys Leu Ile Ser Gly Trp Gly Asn Thr Lys Ser Ser Gly
115 120 125
Ser Ser Tyr Pro Ser Leu Leu Gln Cys Leu Lys Ala Pro Val Leu Ser
130 135 140
Asp Ser Ser Cys Lys Ser Ser Tyr Pro Gly Gln Ile Thr Gly Asn Met
145 150 155 160
Ile Cys Val Gly Phe Leu Glu Gly Gly Lys Asp Ala Cys Gln Gly Asp
165 170 175
Ser Gly Gly Pro Val Val Cys Asn Gly Gln Leu Gln Gly Ile Val Ser
180 185 190
Ser Gly Tyr Gly Cys Ala Gln Lys Asn Lys Pro Gly Val Tyr Thr Lys
195 200 205
Val Cys Asn Tyr Val Asn Trp Ile Gln Gln Thr Ile Ala Ala Asn
210 215 220
<210> 48
<211> 223
<212> PRT
<213> 人工序列
<220>
<223> 猪胰蛋白酶变体48号
<400> 48
Ile Val Gly Gly Tyr Thr Cys Ala Ala Asn Ser Ile Pro Tyr Gln Val
1 5 10 15
Ser Leu Asn Ser Gly Ser His Met Cys Gly Gly Ser Leu Ile Asn Ser
20 25 30
Gln Trp Val Val Ser Ala Ala His Cys Tyr Lys Ser Arg Ile Gln Val
35 40 45
Arg Leu Gly Glu His Asn Ile Asp Val Leu Glu Gly Asn Glu Gln Phe
50 55 60
Ile Asn Ala Ala Lys Ile Ile Thr His Pro Asn Phe Asn Gly Asn Thr
65 70 75 80
Leu Asp Asn Asp Ile Met Leu Ile Lys Leu Ser Ser Pro Ala Thr Leu
85 90 95
Asn Ser Arg Val Ala Thr Val Ser Leu Pro Arg Ser Cys Ala Ala Ala
100 105 110
Gly Thr Glu Cys Leu Ile Ser Gly Trp Gly Asn Thr Lys Ser Ser Gly
115 120 125
Ser Ser Tyr Pro Ser Leu Leu Gln Cys Leu Lys Ala Pro Val Leu Ser
130 135 140
Asp Ser Ser Cys Lys Ser Ser Tyr Pro Gly Gln Ile Thr Gly Asn Met
145 150 155 160
Ile Cys Val Gly Phe Leu Glu Gly Gly Lys Asp Ala Cys Gln Gly Asp
165 170 175
Ser Gly Gly Pro Val Val Cys Asn Gly Gln Leu Gln Gly Ile Val Ser
180 185 190
Ser Gly Tyr Gly Cys Ala Gln Lys Asn Lys Pro Gly Val Tyr Thr Lys
195 200 205
Val Cys Asn Tyr Val Asn Trp Ile Gln Gln Thr Ile Ala Ala Asn
210 215 220
<210> 49
<211> 223
<212> PRT
<213> 人工序列
<220>
<223> 猪胰蛋白酶变体49号
<400> 49
Ile Val Gly Gly Tyr Thr Cys Ala Ala Asn Ser Ile Pro Tyr Gln Val
1 5 10 15
Ser Leu Asn Ser Gly Ser His Thr Cys Gly Gly Ser Leu Ile Asn Ser
20 25 30
Gln Trp Val Val Ser Ala Ala His Cys Tyr Lys Ser Arg Ile Gln Val
35 40 45
Arg Leu Gly Glu His Asn Ile Asp Val Leu Glu Gly Asn Glu Gln Phe
50 55 60
Ile Asn Ala Ala Lys Ile Ile Thr His Pro Asn Phe Asn Gly Asn Thr
65 70 75 80
Leu Asp Asn Asp Ile Met Leu Ile Lys Leu Ser Ser Pro Ala Thr Leu
85 90 95
Asn Ser Arg Val Ala Thr Val Ser Leu Pro Arg Ser Cys Ala Ala Ala
100 105 110
Gly Thr Glu Cys Leu Ile Ser Gly Trp Gly Asn Thr Lys Ser Ser Gly
115 120 125
Ser Ser Tyr Pro Ser Leu Leu Gln Cys Leu Lys Ala Pro Val Leu Ser
130 135 140
Asp Ser Ser Cys Lys Ser Ser Tyr Pro Gly Gln Ile Thr Gly Asn Met
145 150 155 160
Ile Cys Val Gly Phe Leu Glu Gly Gly Lys Asp Ala Cys Gln Gly Asp
165 170 175
Ser Gly Gly Pro Val Val Cys Asn Gly Gln Leu Gln Gly Ile Val Ser
180 185 190
Ser Gly Tyr Gly Cys Ala Gln Lys Asn Lys Pro Gly Val Tyr Thr Lys
195 200 205
Val Cys Asn Tyr Val Asn Trp Ile Gln Gln Thr Ile Ala Ala Asn
210 215 220
<210> 50
<211> 223
<212> PRT
<213> 人工序列
<220>
<223> 猪胰蛋白酶变体50号
<400> 50
Ile Val Gly Gly Tyr Thr Cys Ala Ala Asn Ser Ile Pro Tyr Gln Val
1 5 10 15
Ser Leu Asn Ser Gly Ser His Asn Cys Gly Gly Ser Leu Ile Asn Ser
20 25 30
Gln Trp Val Val Ser Ala Ala His Cys Tyr Lys Ser Arg Ile Gln Val
35 40 45
Arg Leu Gly Glu His Asn Ile Asp Val Leu Glu Gly Asn Glu Gln Phe
50 55 60
Ile Asn Ala Ala Lys Ile Ile Thr His Pro Asn Phe Asn Gly Asn Thr
65 70 75 80
Leu Asp Asn Asp Ile Met Leu Ile Lys Leu Ser Ser Pro Ala Thr Leu
85 90 95
Asn Ser Arg Val Ala Thr Val Ser Leu Pro Arg Ser Cys Ala Ala Ala
100 105 110
Gly Thr Glu Cys Leu Ile Ser Gly Trp Gly Asn Thr Lys Ser Ser Gly
115 120 125
Ser Ser Tyr Pro Ser Leu Leu Gln Cys Leu Lys Ala Pro Val Leu Ser
130 135 140
Asp Ser Ser Cys Lys Ser Ser Tyr Pro Gly Gln Ile Thr Gly Asn Met
145 150 155 160
Ile Cys Val Gly Phe Leu Glu Gly Gly Lys Asp Ala Cys Gln Gly Asp
165 170 175
Ser Gly Gly Pro Val Val Cys Asn Gly Gln Leu Gln Gly Ile Val Ser
180 185 190
Ser Gly Tyr Gly Cys Ala Gln Lys Asn Lys Pro Gly Val Tyr Thr Lys
195 200 205
Val Cys Asn Tyr Val Asn Trp Ile Gln Gln Thr Ile Ala Ala Asn
210 215 220
<210> 51
<211> 223
<212> PRT
<213> 人工序列
<220>
<223> 猪胰蛋白酶变体51号
<400> 51
Ile Val Gly Gly Tyr Thr Cys Ala Ala Asn Ser Ile Pro Tyr Gln Val
1 5 10 15
Ser Leu Asn Ser Gly Ser His Lys Cys Gly Gly Ser Leu Ile Asn Ser
20 25 30
Gln Trp Val Val Ser Ala Ala His Cys Tyr Lys Ser Arg Ile Gln Val
35 40 45
Arg Leu Gly Glu His Asn Ile Asp Val Leu Glu Gly Asn Glu Gln Phe
50 55 60
Ile Asn Ala Ala Lys Ile Ile Thr His Pro Asn Phe Asn Gly Asn Thr
65 70 75 80
Leu Asp Asn Asp Ile Met Leu Ile Lys Leu Ser Ser Pro Ala Thr Leu
85 90 95
Asn Ser Arg Val Ala Thr Val Ser Leu Pro Arg Ser Cys Ala Ala Ala
100 105 110
Gly Thr Glu Cys Leu Ile Ser Gly Trp Gly Asn Thr Lys Ser Ser Gly
115 120 125
Ser Ser Tyr Pro Ser Leu Leu Gln Cys Leu Lys Ala Pro Val Leu Ser
130 135 140
Asp Ser Ser Cys Lys Ser Ser Tyr Pro Gly Gln Ile Thr Gly Asn Met
145 150 155 160
Ile Cys Val Gly Phe Leu Glu Gly Gly Lys Asp Ala Cys Gln Gly Asp
165 170 175
Ser Gly Gly Pro Val Val Cys Asn Gly Gln Leu Gln Gly Ile Val Ser
180 185 190
Ser Gly Tyr Gly Cys Ala Gln Lys Asn Lys Pro Gly Val Tyr Thr Lys
195 200 205
Val Cys Asn Tyr Val Asn Trp Ile Gln Gln Thr Ile Ala Ala Asn
210 215 220
<210> 52
<211> 223
<212> PRT
<213> 人工序列
<220>
<223> 猪胰蛋白酶变体52号
<400> 52
Ile Val Gly Gly Tyr Thr Cys Ala Ala Asn Ser Ile Pro Tyr Gln Val
1 5 10 15
Ser Leu Asn Ser Gly Ser His Arg Cys Gly Gly Ser Leu Ile Asn Ser
20 25 30
Gln Trp Val Val Ser Ala Ala His Cys Tyr Lys Leu Arg Ile Gln Val
35 40 45
Arg Leu Gly Glu His Asn Ile Asp Val Leu Glu Gly Asn Glu Gln Phe
50 55 60
Ile Asn Ala Ala Lys Ile Ile Thr His Pro Asn Phe Asn Gly Asn Thr
65 70 75 80
Leu Asp Asn Asp Ile Met Leu Ile Lys Leu Ser Ser Pro Ala Thr Leu
85 90 95
Asn Ser Arg Val Ala Thr Val Ser Leu Pro Arg Ser Cys Ala Ala Ala
100 105 110
Gly Thr Glu Cys Leu Ile Ser Gly Trp Gly Asn Thr Lys Ser Ser Gly
115 120 125
Ser Ser Tyr Pro Ser Leu Leu Gln Cys Leu Lys Ala Pro Val Leu Ser
130 135 140
Asp Ser Ser Cys Lys Ser Ser Tyr Pro Gly Gln Ile Thr Gly Asn Met
145 150 155 160
Ile Cys Val Gly Phe Leu Glu Gly Gly Lys Asp Ala Cys Gln Gly Asp
165 170 175
Ser Gly Gly Pro Val Val Cys Asn Gly Gln Leu Gln Gly Ile Val Ser
180 185 190
Ser Gly Tyr Gly Cys Ala Gln Lys Asn Lys Pro Gly Val Tyr Thr Lys
195 200 205
Val Cys Asn Tyr Val Asn Trp Ile Gln Gln Thr Ile Ala Ala Asn
210 215 220
<210> 53
<211> 223
<212> PRT
<213> 人工序列
<220>
<223> 猪胰蛋白酶变体53号
<400> 53
Ile Val Gly Gly Tyr Thr Cys Ala Ala Asn Ser Ile Pro Tyr Gln Val
1 5 10 15
Ser Leu Asn Ser Gly Ser His Arg Cys Gly Gly Ser Leu Ile Asn Ser
20 25 30
Gln Trp Val Val Ser Ala Ala His Cys Tyr Lys Pro Arg Ile Gln Val
35 40 45
Arg Leu Gly Glu His Asn Ile Asp Val Leu Glu Gly Asn Glu Gln Phe
50 55 60
Ile Asn Ala Ala Lys Ile Ile Thr His Pro Asn Phe Asn Gly Asn Thr
65 70 75 80
Leu Asp Asn Asp Ile Met Leu Ile Lys Leu Ser Ser Pro Ala Thr Leu
85 90 95
Asn Ser Arg Val Ala Thr Val Ser Leu Pro Arg Ser Cys Ala Ala Ala
100 105 110
Gly Thr Glu Cys Leu Ile Ser Gly Trp Gly Asn Thr Lys Ser Ser Gly
115 120 125
Ser Ser Tyr Pro Ser Leu Leu Gln Cys Leu Lys Ala Pro Val Leu Ser
130 135 140
Asp Ser Ser Cys Lys Ser Ser Tyr Pro Gly Gln Ile Thr Gly Asn Met
145 150 155 160
Ile Cys Val Gly Phe Leu Glu Gly Gly Lys Asp Ala Cys Gln Gly Asp
165 170 175
Ser Gly Gly Pro Val Val Cys Asn Gly Gln Leu Gln Gly Ile Val Ser
180 185 190
Ser Gly Tyr Gly Cys Ala Gln Lys Asn Lys Pro Gly Val Tyr Thr Lys
195 200 205
Val Cys Asn Tyr Val Asn Trp Ile Gln Gln Thr Ile Ala Ala Asn
210 215 220
<210> 54
<211> 223
<212> PRT
<213> 人工序列
<220>
<223> 猪胰蛋白酶变体54号
<400> 54
Ile Val Gly Gly Tyr Thr Cys Ala Ala Asn Ser Ile Pro Tyr Gln Val
1 5 10 15
Ser Leu Asn Ser Gly Ser His Arg Cys Gly Gly Ser Leu Ile Asn Ser
20 25 30
Gln Trp Val Val Ser Ala Ala His Cys Tyr Lys Ser Arg Ile Gln Val
35 40 45
Arg Leu Gly Glu His Asn Ile Ala Val Leu Glu Gly Asn Glu Gln Phe
50 55 60
Ile Asn Ala Ala Lys Ile Ile Thr His Pro Asn Phe Asn Gly Asn Thr
65 70 75 80
Leu Asp Asn Asp Ile Met Leu Ile Lys Leu Ser Ser Pro Ala Thr Leu
85 90 95
Asn Ser Arg Val Ala Thr Val Ser Leu Pro Arg Ser Cys Ala Ala Ala
100 105 110
Gly Thr Glu Cys Leu Ile Ser Gly Trp Gly Asn Thr Lys Ser Ser Gly
115 120 125
Ser Ser Tyr Pro Ser Leu Leu Gln Cys Leu Lys Ala Pro Val Leu Ser
130 135 140
Asp Ser Ser Cys Lys Ser Ser Tyr Pro Gly Gln Ile Thr Gly Asn Met
145 150 155 160
Ile Cys Val Gly Phe Leu Glu Gly Gly Lys Asp Ala Cys Gln Gly Asp
165 170 175
Ser Gly Gly Pro Val Val Cys Asn Gly Gln Leu Gln Gly Ile Val Ser
180 185 190
Ser Gly Tyr Gly Cys Ala Gln Lys Asn Lys Pro Gly Val Tyr Thr Lys
195 200 205
Val Cys Asn Tyr Val Asn Trp Ile Gln Gln Thr Ile Ala Ala Asn
210 215 220
<210> 55
<211> 223
<212> PRT
<213> 人工序列
<220>
<223> 猪胰蛋白酶变体55号
<400> 55
Ile Val Gly Gly Tyr Thr Cys Ala Ala Asn Ser Ile Pro Tyr Gln Val
1 5 10 15
Ser Leu Asn Ser Gly Ser His Arg Cys Gly Gly Ser Leu Ile Asn Ser
20 25 30
Gln Trp Val Val Ser Ala Ala His Cys Tyr Lys Ser Arg Ile Gln Val
35 40 45
Arg Leu Gly Glu His Asn Ile Trp Val Leu Glu Gly Asn Glu Gln Phe
50 55 60
Ile Asn Ala Ala Lys Ile Ile Thr His Pro Asn Phe Asn Gly Asn Thr
65 70 75 80
Leu Asp Asn Asp Ile Met Leu Ile Lys Leu Ser Ser Pro Ala Thr Leu
85 90 95
Asn Ser Arg Val Ala Thr Val Ser Leu Pro Arg Ser Cys Ala Ala Ala
100 105 110
Gly Thr Glu Cys Leu Ile Ser Gly Trp Gly Asn Thr Lys Ser Ser Gly
115 120 125
Ser Ser Tyr Pro Ser Leu Leu Gln Cys Leu Lys Ala Pro Val Leu Ser
130 135 140
Asp Ser Ser Cys Lys Ser Ser Tyr Pro Gly Gln Ile Thr Gly Asn Met
145 150 155 160
Ile Cys Val Gly Phe Leu Glu Gly Gly Lys Asp Ala Cys Gln Gly Asp
165 170 175
Ser Gly Gly Pro Val Val Cys Asn Gly Gln Leu Gln Gly Ile Val Ser
180 185 190
Ser Gly Tyr Gly Cys Ala Gln Lys Asn Lys Pro Gly Val Tyr Thr Lys
195 200 205
Val Cys Asn Tyr Val Asn Trp Ile Gln Gln Thr Ile Ala Ala Asn
210 215 220
<210> 56
<211> 223
<212> PRT
<213> 人工序列
<220>
<223> 猪胰蛋白酶变体56号
<400> 56
Ile Val Gly Gly Tyr Thr Cys Ala Ala Asn Ser Ile Pro Tyr Gln Val
1 5 10 15
Ser Leu Asn Ser Gly Ser His Arg Cys Gly Gly Ser Leu Ile Asn Ser
20 25 30
Gln Trp Val Val Ser Ala Ala His Cys Tyr Lys Ser Arg Ile Gln Val
35 40 45
Arg Leu Gly Glu His Asn Ile Asn Val Leu Glu Gly Asn Glu Gln Phe
50 55 60
Ile Asn Ala Ala Lys Ile Ile Thr His Pro Asn Phe Asn Gly Asn Thr
65 70 75 80
Leu Asp Asn Asp Ile Met Leu Ile Lys Leu Ser Ser Pro Ala Thr Leu
85 90 95
Asn Ser Arg Val Ala Thr Val Ser Leu Pro Arg Ser Cys Ala Ala Ala
100 105 110
Gly Thr Glu Cys Leu Ile Ser Gly Trp Gly Asn Thr Lys Ser Ser Gly
115 120 125
Ser Ser Tyr Pro Ser Leu Leu Gln Cys Leu Lys Ala Pro Val Leu Ser
130 135 140
Asp Ser Ser Cys Lys Ser Ser Tyr Pro Gly Gln Ile Thr Gly Asn Met
145 150 155 160
Ile Cys Val Gly Phe Leu Glu Gly Gly Lys Asp Ala Cys Gln Gly Asp
165 170 175
Ser Gly Gly Pro Val Val Cys Asn Gly Gln Leu Gln Gly Ile Val Ser
180 185 190
Ser Gly Tyr Gly Cys Ala Gln Lys Asn Lys Pro Gly Val Tyr Thr Lys
195 200 205
Val Cys Asn Tyr Val Asn Trp Ile Gln Gln Thr Ile Ala Ala Asn
210 215 220
<210> 57
<211> 223
<212> PRT
<213> 人工序列
<220>
<223> 猪胰蛋白酶变体57号
<400> 57
Ile Val Gly Gly Tyr Thr Cys Ala Ala Asn Ser Ile Pro Tyr Gln Val
1 5 10 15
Ser Leu Asn Ser Gly Ser His Arg Cys Gly Gly Ser Leu Ile Asn Ser
20 25 30
Gln Trp Val Val Ser Ala Ala His Cys Tyr Lys Ser Arg Ile Gln Val
35 40 45
Arg Leu Gly Glu His Asn Ile His Val Leu Glu Gly Asn Glu Gln Phe
50 55 60
Ile Asn Ala Ala Lys Ile Ile Thr His Pro Asn Phe Asn Gly Asn Thr
65 70 75 80
Leu Asp Asn Asp Ile Met Leu Ile Lys Leu Ser Ser Pro Ala Thr Leu
85 90 95
Asn Ser Arg Val Ala Thr Val Ser Leu Pro Arg Ser Cys Ala Ala Ala
100 105 110
Gly Thr Glu Cys Leu Ile Ser Gly Trp Gly Asn Thr Lys Ser Ser Gly
115 120 125
Ser Ser Tyr Pro Ser Leu Leu Gln Cys Leu Lys Ala Pro Val Leu Ser
130 135 140
Asp Ser Ser Cys Lys Ser Ser Tyr Pro Gly Gln Ile Thr Gly Asn Met
145 150 155 160
Ile Cys Val Gly Phe Leu Glu Gly Gly Lys Asp Ala Cys Gln Gly Asp
165 170 175
Ser Gly Gly Pro Val Val Cys Asn Gly Gln Leu Gln Gly Ile Val Ser
180 185 190
Ser Gly Tyr Gly Cys Ala Gln Lys Asn Lys Pro Gly Val Tyr Thr Lys
195 200 205
Val Cys Asn Tyr Val Asn Trp Ile Gln Gln Thr Ile Ala Ala Asn
210 215 220
<210> 58
<211> 223
<212> PRT
<213> 人工序列
<220>
<223> 猪胰蛋白酶变体58号
<400> 58
Ile Val Gly Gly Tyr Thr Cys Ala Ala Asn Ser Ile Pro Tyr Gln Val
1 5 10 15
Ser Leu Asn Ser Gly Ser His Arg Cys Gly Gly Ser Leu Ile Asn Ser
20 25 30
Gln Trp Val Val Ser Ala Ala His Cys Tyr Lys Ser Arg Ile Gln Val
35 40 45
Arg Leu Gly Glu His Asn Ile Asp Val Leu Glu Gly Asn Glu Gln Phe
50 55 60
Ile Asn Ala Ala Lys Ile Ile Thr His Pro Asn Phe Asn Gly Asn Thr
65 70 75 80
Leu Asp Asn Asp Ile Met Leu Ile Lys Leu Ser Ser Pro Ala Thr Leu
85 90 95
Asn Ser Arg Val Ala Thr Val Ser Leu Pro Pro Ser Cys Ala Ala Ala
100 105 110
Gly Thr Glu Cys Leu Ile Ser Gly Trp Gly Asn Thr Lys Ser Ser Gly
115 120 125
Ser Ser Tyr Pro Ser Leu Leu Gln Cys Leu Lys Ala Pro Val Leu Ser
130 135 140
Asp Ser Ser Cys Lys Ser Ser Tyr Pro Gly Gln Ile Thr Gly Asn Met
145 150 155 160
Ile Cys Val Gly Phe Leu Glu Gly Gly Lys Asp Ala Cys Gln Gly Asp
165 170 175
Ser Gly Gly Pro Val Val Cys Asn Gly Gln Leu Gln Gly Ile Val Ser
180 185 190
Ser Gly Tyr Gly Cys Ala Gln Lys Asn Lys Pro Gly Val Tyr Thr Lys
195 200 205
Val Cys Asn Tyr Val Asn Trp Ile Gln Gln Thr Ile Ala Ala Asn
210 215 220
<210> 59
<211> 223
<212> PRT
<213> 人工序列
<220>
<223> 猪胰蛋白酶变体59号
<400> 59
Ile Val Gly Gly Tyr Thr Cys Ala Ala Asn Ser Ile Pro Tyr Gln Val
1 5 10 15
Ser Leu Asn Ser Gly Ser His Arg Cys Gly Gly Ser Leu Ile Asn Ser
20 25 30
Gln Trp Val Val Ser Ala Ala His Cys Tyr Lys Ser Arg Ile Gln Val
35 40 45
Arg Leu Gly Glu His Asn Ile Asp Val Leu Glu Gly Asn Glu Gln Phe
50 55 60
Ile Asn Ala Ala Lys Ile Ile Thr His Pro Asn Phe Asn Gly Asn Thr
65 70 75 80
Leu Asp Asn Asp Ile Met Leu Ile Lys Leu Ser Ser Pro Ala Thr Leu
85 90 95
Asn Ser Arg Val Ala Thr Val Ser Leu Pro Gly Ser Cys Ala Ala Ala
100 105 110
Gly Thr Glu Cys Leu Ile Ser Gly Trp Gly Asn Thr Lys Ser Ser Gly
115 120 125
Ser Ser Tyr Pro Ser Leu Leu Gln Cys Leu Lys Ala Pro Val Leu Ser
130 135 140
Asp Ser Ser Cys Lys Ser Ser Tyr Pro Gly Gln Ile Thr Gly Asn Met
145 150 155 160
Ile Cys Val Gly Phe Leu Glu Gly Gly Lys Asp Ala Cys Gln Gly Asp
165 170 175
Ser Gly Gly Pro Val Val Cys Asn Gly Gln Leu Gln Gly Ile Val Ser
180 185 190
Ser Gly Tyr Gly Cys Ala Gln Lys Asn Lys Pro Gly Val Tyr Thr Lys
195 200 205
Val Cys Asn Tyr Val Asn Trp Ile Gln Gln Thr Ile Ala Ala Asn
210 215 220
<210> 60
<211> 223
<212> PRT
<213> 人工序列
<220>
<223> 猪胰蛋白酶变体60号
<400> 60
Ile Val Gly Gly Tyr Thr Cys Ala Ala Asn Ser Ile Pro Tyr Gln Val
1 5 10 15
Ser Leu Asn Ser Gly Ser His Arg Cys Gly Gly Ser Leu Ile Asn Ser
20 25 30
Gln Trp Val Val Ser Ala Ala His Cys Tyr Lys Ser Arg Ile Gln Val
35 40 45
Arg Leu Gly Glu His Asn Ile Asp Val Leu Glu Gly Asn Glu Gln Phe
50 55 60
Ile Asn Ala Ala Lys Ile Ile Thr His Pro Asn Phe Asn Gly Asn Thr
65 70 75 80
Leu Asp Asn Asp Ile Met Leu Ile Lys Leu Ser Ser Pro Ala Thr Leu
85 90 95
Asn Ser Arg Val Ala Thr Val Ser Leu Pro Ser Ser Cys Ala Ala Ala
100 105 110
Gly Thr Glu Cys Leu Ile Ser Gly Trp Gly Asn Thr Lys Ser Ser Gly
115 120 125
Ser Ser Tyr Pro Ser Leu Leu Gln Cys Leu Lys Ala Pro Val Leu Ser
130 135 140
Asp Ser Ser Cys Lys Ser Ser Tyr Pro Gly Gln Ile Thr Gly Asn Met
145 150 155 160
Ile Cys Val Gly Phe Leu Glu Gly Gly Lys Asp Ala Cys Gln Gly Asp
165 170 175
Ser Gly Gly Pro Val Val Cys Asn Gly Gln Leu Gln Gly Ile Val Ser
180 185 190
Ser Gly Tyr Gly Cys Ala Gln Lys Asn Lys Pro Gly Val Tyr Thr Lys
195 200 205
Val Cys Asn Tyr Val Asn Trp Ile Gln Gln Thr Ile Ala Ala Asn
210 215 220
<210> 61
<211> 223
<212> PRT
<213> 人工序列
<220>
<223> 猪胰蛋白酶变体61号
<400> 61
Ile Val Gly Gly Tyr Thr Cys Ala Ala Asn Ser Ile Pro Tyr Gln Val
1 5 10 15
Ser Leu Asn Ser Gly Ser His Arg Cys Gly Gly Ser Leu Ile Asn Ser
20 25 30
Gln Trp Val Val Ser Ala Ala His Cys Tyr Lys Ser Arg Ile Gln Val
35 40 45
Arg Leu Gly Glu His Asn Ile Asp Val Leu Glu Gly Asn Glu Gln Phe
50 55 60
Ile Asn Ala Ala Lys Ile Ile Thr His Pro Asn Phe Asn Gly Asn Thr
65 70 75 80
Leu Asp Asn Asp Ile Met Leu Ile Lys Leu Ser Ser Pro Ala Thr Leu
85 90 95
Asn Ser Arg Val Ala Thr Val Ser Leu Pro Asp Ser Cys Ala Ala Ala
100 105 110
Gly Thr Glu Cys Leu Ile Ser Gly Trp Gly Asn Thr Lys Ser Ser Gly
115 120 125
Ser Ser Tyr Pro Ser Leu Leu Gln Cys Leu Lys Ala Pro Val Leu Ser
130 135 140
Asp Ser Ser Cys Lys Ser Ser Tyr Pro Gly Gln Ile Thr Gly Asn Met
145 150 155 160
Ile Cys Val Gly Phe Leu Glu Gly Gly Lys Asp Ala Cys Gln Gly Asp
165 170 175
Ser Gly Gly Pro Val Val Cys Asn Gly Gln Leu Gln Gly Ile Val Ser
180 185 190
Ser Gly Tyr Gly Cys Ala Gln Lys Asn Lys Pro Gly Val Tyr Thr Lys
195 200 205
Val Cys Asn Tyr Val Asn Trp Ile Gln Gln Thr Ile Ala Ala Asn
210 215 220
<210> 62
<211> 223
<212> PRT
<213> 人工序列
<220>
<223> 猪胰蛋白酶变体62号
<400> 62
Ile Val Gly Gly Tyr Thr Cys Ala Ala Asn Ser Ile Pro Tyr Gln Val
1 5 10 15
Ser Leu Asn Ser Gly Ser His Arg Cys Gly Gly Ser Leu Ile Asn Ser
20 25 30
Gln Trp Val Val Ser Ala Ala His Cys Tyr Lys Ser Arg Ile Gln Val
35 40 45
Arg Leu Gly Glu His Asn Ile Asp Val Leu Glu Gly Asn Glu Gln Phe
50 55 60
Ile Asn Ala Ala Lys Ile Ile Thr His Pro Asn Phe Asn Gly Asn Thr
65 70 75 80
Leu Asp Asn Asp Ile Met Leu Ile Lys Leu Ser Ser Pro Ala Thr Leu
85 90 95
Asn Ser Arg Val Ala Thr Val Ser Leu Pro Arg Ser Cys Ala Ala Ala
100 105 110
Gly Thr Glu Cys Leu Ile Ser Gly Trp Gly Asn Thr Lys Ser Ser Gly
115 120 125
Ser Ser Val Pro Ser Leu Leu Gln Cys Leu Lys Ala Pro Val Leu Ser
130 135 140
Asp Ser Ser Cys Lys Ser Ser Tyr Pro Gly Gln Ile Thr Gly Asn Met
145 150 155 160
Ile Cys Val Gly Phe Leu Glu Gly Gly Lys Asp Ala Cys Gln Gly Asp
165 170 175
Ser Gly Gly Pro Val Val Cys Asn Gly Gln Leu Gln Gly Ile Val Ser
180 185 190
Ser Gly Tyr Gly Cys Ala Gln Lys Asn Lys Pro Gly Val Tyr Thr Lys
195 200 205
Val Cys Asn Tyr Val Asn Trp Ile Gln Gln Thr Ile Ala Ala Asn
210 215 220
<210> 63
<211> 223
<212> PRT
<213> 人工序列
<220>
<223> 猪胰蛋白酶变体63号
<400> 63
Ile Val Gly Gly Tyr Thr Cys Ala Ala Asn Ser Ile Pro Tyr Gln Val
1 5 10 15
Ser Leu Asn Ser Gly Ser His Arg Cys Gly Gly Ser Leu Ile Asn Ser
20 25 30
Gln Trp Val Val Ser Ala Ala His Cys Tyr Lys Ser Arg Ile Gln Val
35 40 45
Arg Leu Gly Glu His Asn Ile Asp Val Leu Glu Gly Asn Glu Gln Phe
50 55 60
Ile Asn Ala Ala Lys Ile Ile Thr His Pro Asn Phe Asn Gly Asn Thr
65 70 75 80
Leu Asp Asn Asp Ile Met Leu Ile Lys Leu Ser Ser Pro Ala Thr Leu
85 90 95
Asn Ser Arg Val Ala Thr Val Ser Leu Pro Arg Ser Cys Ala Ala Ala
100 105 110
Gly Thr Glu Cys Leu Ile Ser Gly Trp Gly Asn Thr Lys Ser Ser Gly
115 120 125
Ser Ser Ile Pro Ser Leu Leu Gln Cys Leu Lys Ala Pro Val Leu Ser
130 135 140
Asp Ser Ser Cys Lys Ser Ser Tyr Pro Gly Gln Ile Thr Gly Asn Met
145 150 155 160
Ile Cys Val Gly Phe Leu Glu Gly Gly Lys Asp Ala Cys Gln Gly Asp
165 170 175
Ser Gly Gly Pro Val Val Cys Asn Gly Gln Leu Gln Gly Ile Val Ser
180 185 190
Ser Gly Tyr Gly Cys Ala Gln Lys Asn Lys Pro Gly Val Tyr Thr Lys
195 200 205
Val Cys Asn Tyr Val Asn Trp Ile Gln Gln Thr Ile Ala Ala Asn
210 215 220
<210> 64
<211> 223
<212> PRT
<213> 人工序列
<220>
<223> 猪胰蛋白酶变体64号
<400> 64
Ile Val Gly Gly Tyr Thr Cys Ala Ala Asn Ser Ile Pro Tyr Gln Val
1 5 10 15
Ser Leu Asn Ser Gly Ser His Arg Cys Gly Gly Ser Leu Ile Asn Ser
20 25 30
Gln Trp Val Val Ser Ala Ala His Cys Tyr Lys Ser Arg Ile Gln Val
35 40 45
Arg Leu Gly Glu His Asn Ile Asp Val Leu Glu Gly Asn Glu Gln Phe
50 55 60
Ile Asn Ala Ala Lys Ile Ile Thr His Pro Asn Phe Asn Gly Asn Thr
65 70 75 80
Leu Asp Asn Asp Ile Met Leu Ile Lys Leu Ser Ser Pro Ala Thr Leu
85 90 95
Asn Ser Arg Val Ala Thr Val Ser Leu Pro Arg Ser Cys Ala Ala Ala
100 105 110
Gly Thr Glu Cys Leu Ile Ser Gly Trp Gly Asn Thr Lys Ser Ser Gly
115 120 125
Ser Ser Trp Pro Ser Leu Leu Gln Cys Leu Lys Ala Pro Val Leu Ser
130 135 140
Asp Ser Ser Cys Lys Ser Ser Tyr Pro Gly Gln Ile Thr Gly Asn Met
145 150 155 160
Ile Cys Val Gly Phe Leu Glu Gly Gly Lys Asp Ala Cys Gln Gly Asp
165 170 175
Ser Gly Gly Pro Val Val Cys Asn Gly Gln Leu Gln Gly Ile Val Ser
180 185 190
Ser Gly Tyr Gly Cys Ala Gln Lys Asn Lys Pro Gly Val Tyr Thr Lys
195 200 205
Val Cys Asn Tyr Val Asn Trp Ile Gln Gln Thr Ile Ala Ala Asn
210 215 220
<210> 65
<211> 223
<212> PRT
<213> 人工序列
<220>
<223> 猪胰蛋白酶变体65号
<400> 65
Ile Val Gly Gly Tyr Thr Cys Ala Ala Asn Ser Ile Pro Tyr Gln Val
1 5 10 15
Ser Leu Asn Ser Gly Ser His Arg Cys Gly Gly Ser Leu Ile Asn Ser
20 25 30
Gln Trp Val Val Ser Ala Ala His Cys Tyr Lys Ser Arg Ile Gln Val
35 40 45
Arg Leu Gly Glu His Asn Ile Asp Val Leu Glu Gly Asn Glu Gln Phe
50 55 60
Ile Asn Ala Ala Lys Ile Ile Thr His Pro Asn Phe Asn Gly Asn Thr
65 70 75 80
Leu Asp Asn Asp Ile Met Leu Ile Lys Leu Ser Ser Pro Ala Thr Leu
85 90 95
Asn Ser Arg Val Ala Thr Val Ser Leu Pro Arg Ser Cys Ala Ala Ala
100 105 110
Gly Thr Glu Cys Leu Ile Ser Gly Trp Gly Asn Thr Lys Ser Ser Gly
115 120 125
Ser Ser Gly Pro Ser Leu Leu Gln Cys Leu Lys Ala Pro Val Leu Ser
130 135 140
Asp Ser Ser Cys Lys Ser Ser Tyr Pro Gly Gln Ile Thr Gly Asn Met
145 150 155 160
Ile Cys Val Gly Phe Leu Glu Gly Gly Lys Asp Ala Cys Gln Gly Asp
165 170 175
Ser Gly Gly Pro Val Val Cys Asn Gly Gln Leu Gln Gly Ile Val Ser
180 185 190
Ser Gly Tyr Gly Cys Ala Gln Lys Asn Lys Pro Gly Val Tyr Thr Lys
195 200 205
Val Cys Asn Tyr Val Asn Trp Ile Gln Gln Thr Ile Ala Ala Asn
210 215 220
<210> 66
<211> 223
<212> PRT
<213> 人工序列
<220>
<223> 猪胰蛋白酶变体66号
<400> 66
Ile Val Gly Gly Tyr Thr Cys Ala Ala Asn Ser Ile Pro Tyr Gln Val
1 5 10 15
Ser Leu Asn Ser Gly Ser His Arg Cys Gly Gly Ser Leu Ile Asn Ser
20 25 30
Gln Trp Val Val Ser Ala Ala His Cys Tyr Lys Ser Arg Ile Gln Val
35 40 45
Arg Leu Gly Glu His Asn Ile Asp Val Leu Glu Gly Asn Glu Gln Phe
50 55 60
Ile Asn Ala Ala Lys Ile Ile Thr His Pro Asn Phe Asn Gly Asn Thr
65 70 75 80
Leu Asp Asn Asp Ile Met Leu Ile Lys Leu Ser Ser Pro Ala Thr Leu
85 90 95
Asn Ser Arg Val Ala Thr Val Ser Leu Pro Arg Ser Cys Ala Ala Ala
100 105 110
Gly Thr Glu Cys Leu Ile Ser Gly Trp Gly Asn Thr Lys Ser Ser Gly
115 120 125
Ser Ser Ser Pro Ser Leu Leu Gln Cys Leu Lys Ala Pro Val Leu Ser
130 135 140
Asp Ser Ser Cys Lys Ser Ser Tyr Pro Gly Gln Ile Thr Gly Asn Met
145 150 155 160
Ile Cys Val Gly Phe Leu Glu Gly Gly Lys Asp Ala Cys Gln Gly Asp
165 170 175
Ser Gly Gly Pro Val Val Cys Asn Gly Gln Leu Gln Gly Ile Val Ser
180 185 190
Ser Gly Tyr Gly Cys Ala Gln Lys Asn Lys Pro Gly Val Tyr Thr Lys
195 200 205
Val Cys Asn Tyr Val Asn Trp Ile Gln Gln Thr Ile Ala Ala Asn
210 215 220
<210> 67
<211> 223
<212> PRT
<213> 人工序列
<220>
<223> 猪胰蛋白酶变体67号
<400> 67
Ile Val Gly Gly Tyr Thr Cys Ala Ala Asn Ser Ile Pro Tyr Gln Val
1 5 10 15
Ser Leu Asn Ser Gly Ser His Arg Cys Gly Gly Ser Leu Ile Asn Ser
20 25 30
Gln Trp Val Val Ser Ala Ala His Cys Tyr Lys Ser Arg Ile Gln Val
35 40 45
Arg Leu Gly Glu His Asn Ile Asp Val Leu Glu Gly Asn Glu Gln Phe
50 55 60
Ile Asn Ala Ala Lys Ile Ile Thr His Pro Asn Phe Asn Gly Asn Thr
65 70 75 80
Leu Asp Asn Asp Ile Met Leu Ile Lys Leu Ser Ser Pro Ala Thr Leu
85 90 95
Asn Ser Arg Val Ala Thr Val Ser Leu Pro Arg Ser Cys Ala Ala Ala
100 105 110
Gly Thr Glu Cys Leu Ile Ser Gly Trp Gly Asn Thr Lys Ser Ser Gly
115 120 125
Ser Ser Gln Pro Ser Leu Leu Gln Cys Leu Lys Ala Pro Val Leu Ser
130 135 140
Asp Ser Ser Cys Lys Ser Ser Tyr Pro Gly Gln Ile Thr Gly Asn Met
145 150 155 160
Ile Cys Val Gly Phe Leu Glu Gly Gly Lys Asp Ala Cys Gln Gly Asp
165 170 175
Ser Gly Gly Pro Val Val Cys Asn Gly Gln Leu Gln Gly Ile Val Ser
180 185 190
Ser Gly Tyr Gly Cys Ala Gln Lys Asn Lys Pro Gly Val Tyr Thr Lys
195 200 205
Val Cys Asn Tyr Val Asn Trp Ile Gln Gln Thr Ile Ala Ala Asn
210 215 220
<210> 68
<211> 223
<212> PRT
<213> 人工序列
<220>
<223> 猪胰蛋白酶变体68号
<400> 68
Ile Val Gly Gly Tyr Thr Cys Ala Ala Asn Ser Ile Pro Tyr Gln Val
1 5 10 15
Ser Leu Asn Ser Gly Ser His Arg Cys Gly Gly Ser Leu Ile Asn Ser
20 25 30
Gln Trp Val Val Ser Ala Ala His Cys Tyr Lys Ser Arg Ile Gln Val
35 40 45
Arg Leu Gly Glu His Asn Ile Asp Val Leu Glu Gly Asn Glu Gln Phe
50 55 60
Ile Asn Ala Ala Lys Ile Ile Thr His Pro Asn Phe Asn Gly Asn Thr
65 70 75 80
Leu Asp Asn Asp Ile Met Leu Ile Lys Leu Ser Ser Pro Ala Thr Leu
85 90 95
Asn Ser Arg Val Ala Thr Val Ser Leu Pro Arg Ser Cys Ala Ala Ala
100 105 110
Gly Thr Glu Cys Leu Ile Ser Gly Trp Gly Asn Thr Lys Ser Ser Gly
115 120 125
Ser Ser Asp Pro Ser Leu Leu Gln Cys Leu Lys Ala Pro Val Leu Ser
130 135 140
Asp Ser Ser Cys Lys Ser Ser Tyr Pro Gly Gln Ile Thr Gly Asn Met
145 150 155 160
Ile Cys Val Gly Phe Leu Glu Gly Gly Lys Asp Ala Cys Gln Gly Asp
165 170 175
Ser Gly Gly Pro Val Val Cys Asn Gly Gln Leu Gln Gly Ile Val Ser
180 185 190
Ser Gly Tyr Gly Cys Ala Gln Lys Asn Lys Pro Gly Val Tyr Thr Lys
195 200 205
Val Cys Asn Tyr Val Asn Trp Ile Gln Gln Thr Ile Ala Ala Asn
210 215 220
<210> 69
<211> 223
<212> PRT
<213> 人工序列
<220>
<223> 猪胰蛋白酶变体69号
<400> 69
Ile Val Gly Gly Tyr Thr Cys Ala Ala Asn Ser Ile Pro Tyr Gln Val
1 5 10 15
Ser Leu Asn Ser Gly Ser His Arg Cys Gly Gly Ser Leu Ile Asn Ser
20 25 30
Gln Trp Val Val Ser Ala Ala His Cys Tyr Lys Ser Arg Ile Gln Val
35 40 45
Arg Leu Gly Glu His Asn Ile Asp Val Leu Glu Gly Asn Glu Gln Phe
50 55 60
Ile Asn Ala Ala Lys Ile Ile Thr His Pro Asn Phe Asn Gly Asn Thr
65 70 75 80
Leu Asp Asn Asp Ile Met Leu Ile Lys Leu Ser Ser Pro Ala Thr Leu
85 90 95
Asn Ser Arg Val Ala Thr Val Ser Leu Pro Arg Ser Cys Ala Ala Ala
100 105 110
Gly Thr Glu Cys Leu Ile Ser Gly Trp Gly Asn Thr Lys Ser Ser Gly
115 120 125
Ser Ser Tyr Pro Ser Leu Leu Gln Cys Leu Lys Ala Pro Val Leu Ser
130 135 140
Asp Ser Ser Cys Lys Ser Ser Tyr Pro Gly Gln Ile Thr Gly Asn Met
145 150 155 160
Ile Cys Val Gly Phe Leu Glu Gly Gly Lys Asp Ala Cys Gln Gly Asp
165 170 175
Ser Gly Gly Pro Val Val Cys Asn Gly Gln Leu Gln Gly Ile Val Ser
180 185 190
Phe Gly Tyr Gly Cys Ala Gln Lys Asn Lys Pro Gly Val Tyr Thr Lys
195 200 205
Val Cys Asn Tyr Val Asn Trp Ile Gln Gln Thr Ile Ala Ala Asn
210 215 220
<210> 70
<211> 223
<212> PRT
<213> 人工序列
<220>
<223> 猪胰蛋白酶变体70号
<400> 70
Ile Val Gly Gly Tyr Thr Cys Ala Ala Asn Ser Ile Pro Tyr Gln Val
1 5 10 15
Ser Leu Asn Ser Gly Ser His Arg Cys Gly Gly Ser Leu Ile Asn Ser
20 25 30
Gln Trp Val Val Ser Ala Ala His Cys Tyr Lys Ser Arg Ile Gln Val
35 40 45
Arg Leu Gly Glu His Asn Ile Asp Val Leu Glu Gly Asn Glu Gln Phe
50 55 60
Ile Asn Ala Ala Lys Ile Ile Thr His Pro Asn Phe Asn Gly Asn Thr
65 70 75 80
Leu Asp Asn Asp Ile Met Leu Ile Lys Leu Ser Ser Pro Ala Thr Leu
85 90 95
Asn Ser Arg Val Ala Thr Val Ser Leu Pro Arg Ser Cys Ala Ala Ala
100 105 110
Gly Thr Glu Cys Leu Ile Ser Gly Trp Gly Asn Thr Lys Ser Ser Gly
115 120 125
Ser Ser Tyr Pro Ser Leu Leu Gln Cys Leu Lys Ala Pro Val Leu Ser
130 135 140
Asp Ser Ser Cys Lys Ser Ser Tyr Pro Gly Gln Ile Thr Gly Asn Met
145 150 155 160
Ile Cys Val Gly Phe Leu Glu Gly Gly Lys Asp Ala Cys Gln Gly Asp
165 170 175
Ser Gly Gly Pro Val Val Cys Asn Gly Gln Leu Gln Gly Ile Val Ser
180 185 190
Tyr Gly Tyr Gly Cys Ala Gln Lys Asn Lys Pro Gly Val Tyr Thr Lys
195 200 205
Val Cys Asn Tyr Val Asn Trp Ile Gln Gln Thr Ile Ala Ala Asn
210 215 220
<210> 71
<211> 223
<212> PRT
<213> 人工序列
<220>
<223> 猪胰蛋白酶变体71号
<400> 71
Ile Val Gly Gly Tyr Thr Cys Ala Ala Asn Ser Ile Pro Tyr Gln Val
1 5 10 15
Ser Leu Asn Ser Gly Ser His Arg Cys Gly Gly Ser Leu Ile Asn Ser
20 25 30
Gln Trp Val Val Ser Ala Ala His Cys Tyr Lys Ser Arg Ile Gln Val
35 40 45
Arg Leu Gly Glu His Asn Ile Asp Val Leu Glu Gly Asn Glu Gln Phe
50 55 60
Ile Asn Ala Ala Lys Ile Ile Thr His Pro Asn Phe Asn Gly Asn Thr
65 70 75 80
Leu Asp Asn Asp Ile Met Leu Ile Lys Leu Ser Ser Pro Ala Thr Leu
85 90 95
Asn Ser Arg Val Ala Thr Val Ser Leu Pro Arg Ser Cys Ala Ala Ala
100 105 110
Gly Thr Glu Cys Leu Ile Ser Gly Trp Gly Asn Thr Lys Ser Ser Gly
115 120 125
Ser Ser Tyr Pro Ser Leu Leu Gln Cys Leu Lys Ala Pro Val Leu Ser
130 135 140
Asp Ser Ser Cys Lys Ser Ser Tyr Pro Gly Gln Ile Thr Gly Asn Met
145 150 155 160
Ile Cys Val Gly Phe Leu Glu Gly Gly Lys Asp Ala Cys Gln Gly Asp
165 170 175
Ser Gly Gly Pro Val Val Cys Asn Gly Gln Leu Gln Gly Ile Val Ser
180 185 190
Thr Gly Tyr Gly Cys Ala Gln Lys Asn Lys Pro Gly Val Tyr Thr Lys
195 200 205
Val Cys Asn Tyr Val Asn Trp Ile Gln Gln Thr Ile Ala Ala Asn
210 215 220
<210> 72
<211> 223
<212> PRT
<213> 人工序列
<220>
<223> 猪胰蛋白酶变体72号
<400> 72
Ile Val Gly Gly Tyr Thr Cys Ala Ala Asn Ser Ile Pro Tyr Gln Val
1 5 10 15
Ser Leu Asn Ser Gly Ser His Arg Cys Gly Gly Ser Leu Ile Asn Ser
20 25 30
Gln Trp Val Val Ser Ala Ala His Cys Tyr Lys Ser Arg Ile Gln Val
35 40 45
Arg Leu Gly Glu His Asn Ile Asp Val Leu Glu Gly Asn Glu Gln Phe
50 55 60
Ile Asn Ala Ala Lys Ile Ile Thr His Pro Asn Phe Asn Ser Asn Thr
65 70 75 80
Leu Asp Asn Asp Ile Met Leu Ile Lys Leu Ser Ser Pro Ala Thr Leu
85 90 95
Asn Ser Arg Val Ala Thr Val Ser Leu Pro Arg Ser Cys Ala Ala Ala
100 105 110
Gly Thr Glu Cys Leu Ile Ser Gly Trp Gly Asn Thr Lys Ser Ser Gly
115 120 125
Ser Ser Tyr Pro Ser Leu Leu Gln Cys Leu Lys Ala Pro Val Leu Ser
130 135 140
Asp Ser Ser Cys Lys Ser Ser Tyr Pro Gly Gln Ile Thr Gly Asn Met
145 150 155 160
Ile Cys Val Gly Phe Leu Glu Gly Gly Lys Asp Ala Cys Gln Gly Asp
165 170 175
Ser Gly Gly Pro Val Val Cys Asn Gly Gln Leu Gln Gly Ile Val Ser
180 185 190
Ser Gly Tyr Gly Cys Ala Gln Lys Asn Lys Pro Gly Val Tyr Thr Lys
195 200 205
Val Cys Asn Tyr Val Asn Trp Ile Gln Gln Thr Ile Ala Ala Asn
210 215 220
<210> 73
<211> 223
<212> PRT
<213> 人工序列
<220>
<223> 猪胰蛋白酶变体73号
<400> 73
Ile Val Gly Gly Tyr Thr Cys Ala Ala Asn Ser Ile Pro Tyr Gln Val
1 5 10 15
Ser Leu Asn Ser Gly Ser His Arg Cys Gly Gly Ser Leu Ile Asn Ser
20 25 30
Gln Trp Val Val Ser Ala Ala His Cys Tyr Lys Ser Arg Ile Gln Val
35 40 45
Arg Leu Gly Glu His Asn Ile Asp Val Leu Glu Gly Asn Glu Gln Phe
50 55 60
Ile Asn Ala Ala Lys Ile Ile Thr His Pro Asn Phe Asn Ala Asn Thr
65 70 75 80
Leu Asp Asn Asp Ile Met Leu Ile Lys Leu Ser Ser Pro Ala Thr Leu
85 90 95
Asn Ser Arg Val Ala Thr Val Ser Leu Pro Arg Ser Cys Ala Ala Ala
100 105 110
Gly Thr Glu Cys Leu Ile Ser Gly Trp Gly Asn Thr Lys Ser Ser Gly
115 120 125
Ser Ser Tyr Pro Ser Leu Leu Gln Cys Leu Lys Ala Pro Val Leu Ser
130 135 140
Asp Ser Ser Cys Lys Ser Ser Tyr Pro Gly Gln Ile Thr Gly Asn Met
145 150 155 160
Ile Cys Val Gly Phe Leu Glu Gly Gly Lys Asp Ala Cys Gln Gly Asp
165 170 175
Ser Gly Gly Pro Val Val Cys Asn Gly Gln Leu Gln Gly Ile Val Ser
180 185 190
Ser Gly Tyr Gly Cys Ala Gln Lys Asn Lys Pro Gly Val Tyr Thr Lys
195 200 205
Val Cys Asn Tyr Val Asn Trp Ile Gln Gln Thr Ile Ala Ala Asn
210 215 220
<210> 74
<211> 223
<212> PRT
<213> 人工序列
<220>
<223> 猪胰蛋白酶变体74号
<400> 74
Ile Val Gly Gly Tyr Thr Cys Ala Ala Asn Ser Ile Pro Tyr Gln Val
1 5 10 15
Ser Leu Asn Ser Gly Ser His Arg Cys Gly Gly Ser Leu Ile Asn Ser
20 25 30
Gln Trp Val Val Ser Ala Ala His Cys Tyr Lys Ser Arg Ile Gln Val
35 40 45
Arg Leu Gly Glu His Asn Ile Asp Val Leu Glu Gly Asn Glu Gln Phe
50 55 60
Ile Asn Ala Ala Lys Ile Ile Thr His Pro Asn Phe Asn Pro Asn Thr
65 70 75 80
Leu Asp Asn Asp Ile Met Leu Ile Lys Leu Ser Ser Pro Ala Thr Leu
85 90 95
Asn Ser Arg Val Ala Thr Val Ser Leu Pro Arg Ser Cys Ala Ala Ala
100 105 110
Gly Thr Glu Cys Leu Ile Ser Gly Trp Gly Asn Thr Lys Ser Ser Gly
115 120 125
Ser Ser Tyr Pro Ser Leu Leu Gln Cys Leu Lys Ala Pro Val Leu Ser
130 135 140
Asp Ser Ser Cys Lys Ser Ser Tyr Pro Gly Gln Ile Thr Gly Asn Met
145 150 155 160
Ile Cys Val Gly Phe Leu Glu Gly Gly Lys Asp Ala Cys Gln Gly Asp
165 170 175
Ser Gly Gly Pro Val Val Cys Asn Gly Gln Leu Gln Gly Ile Val Ser
180 185 190
Ser Gly Tyr Gly Cys Ala Gln Lys Asn Lys Pro Gly Val Tyr Thr Lys
195 200 205
Val Cys Asn Tyr Val Asn Trp Ile Gln Gln Thr Ile Ala Ala Asn
210 215 220
<210> 75
<211> 223
<212> PRT
<213> 人工序列
<220>
<223> 猪胰蛋白酶变体75号
<400> 75
Ile Val Gly Gly Tyr Thr Cys Ala Ala Asn Ser Ile Pro Tyr Gln Val
1 5 10 15
Ser Leu Asn Ser Gly Ser His Arg Cys Gly Gly Ser Leu Ile Asn Ser
20 25 30
Gln Trp Val Val Ser Ala Ala His Cys Tyr Lys Ser Arg Ile Gln Val
35 40 45
Arg Leu Gly Glu His Asn Ile Asp Val Leu Glu Gly Asn Glu Gln Phe
50 55 60
Ile Asn Ala Ala Lys Ile Ile Thr His Pro Asn Phe Asn Tyr Asn Thr
65 70 75 80
Leu Asp Asn Asp Ile Met Leu Ile Lys Leu Ser Ser Pro Ala Thr Leu
85 90 95
Asn Ser Arg Val Ala Thr Val Ser Leu Pro Arg Ser Cys Ala Ala Ala
100 105 110
Gly Thr Glu Cys Leu Ile Ser Gly Trp Gly Asn Thr Lys Ser Ser Gly
115 120 125
Ser Ser Tyr Pro Ser Leu Leu Gln Cys Leu Lys Ala Pro Val Leu Ser
130 135 140
Asp Ser Ser Cys Lys Ser Ser Tyr Pro Gly Gln Ile Thr Gly Asn Met
145 150 155 160
Ile Cys Val Gly Phe Leu Glu Gly Gly Lys Asp Ala Cys Gln Gly Asp
165 170 175
Ser Gly Gly Pro Val Val Cys Asn Gly Gln Leu Gln Gly Ile Val Ser
180 185 190
Ser Gly Tyr Gly Cys Ala Gln Lys Asn Lys Pro Gly Val Tyr Thr Lys
195 200 205
Val Cys Asn Tyr Val Asn Trp Ile Gln Gln Thr Ile Ala Ala Asn
210 215 220
<210> 76
<211> 223
<212> PRT
<213> 人工序列
<220>
<223> 猪胰蛋白酶变体76号
<400> 76
Ile Val Gly Gly Tyr Thr Cys Ala Ala Asn Ser Ile Pro Tyr Gln Val
1 5 10 15
Ser Leu Asn Ser Gly Ser His Arg Cys Gly Gly Ser Leu Ile Asn Ser
20 25 30
Gln Trp Val Val Ser Ala Ala His Cys Tyr Lys Ser Arg Ile Gln Val
35 40 45
Arg Leu Gly Glu His Asn Ile Asp Val Leu Glu Gly Asn Glu Gln Phe
50 55 60
Ile Asn Ala Ala Lys Ile Ile Thr His Pro Asn Phe Asn Glu Asn Thr
65 70 75 80
Leu Asp Asn Asp Ile Met Leu Ile Lys Leu Ser Ser Pro Ala Thr Leu
85 90 95
Asn Ser Arg Val Ala Thr Val Ser Leu Pro Arg Ser Cys Ala Ala Ala
100 105 110
Gly Thr Glu Cys Leu Ile Ser Gly Trp Gly Asn Thr Lys Ser Ser Gly
115 120 125
Ser Ser Tyr Pro Ser Leu Leu Gln Cys Leu Lys Ala Pro Val Leu Ser
130 135 140
Asp Ser Ser Cys Lys Ser Ser Tyr Pro Gly Gln Ile Thr Gly Asn Met
145 150 155 160
Ile Cys Val Gly Phe Leu Glu Gly Gly Lys Asp Ala Cys Gln Gly Asp
165 170 175
Ser Gly Gly Pro Val Val Cys Asn Gly Gln Leu Gln Gly Ile Val Ser
180 185 190
Ser Gly Tyr Gly Cys Ala Gln Lys Asn Lys Pro Gly Val Tyr Thr Lys
195 200 205
Val Cys Asn Tyr Val Asn Trp Ile Gln Gln Thr Ile Ala Ala Asn
210 215 220
<210> 77
<211> 223
<212> PRT
<213> 人工序列
<220>
<223> 猪胰蛋白酶变体77号
<400> 77
Ile Val Gly Gly Tyr Thr Cys Ala Ala Asn Ser Ile Pro Tyr Gln Val
1 5 10 15
Ser Leu Asn Ser Gly Ser His Arg Cys Gly Gly Ser Leu Ile Asn Ser
20 25 30
Gln Trp Val Val Ser Ala Ala His Cys Tyr Lys Pro Arg Ile Gln Val
35 40 45
Arg Leu Gly Glu His Asn Ile His Val Leu Glu Gly Asn Glu Gln Phe
50 55 60
Ile Asn Ala Ala Lys Ile Ile Thr His Pro Asn Phe Asn Gly Asn Thr
65 70 75 80
Leu Asp Asn Asp Ile Met Leu Ile Lys Leu Ser Ser Pro Ala Thr Leu
85 90 95
Asn Ser His Val Ala Thr Val Ser Leu Pro Thr Ser Cys Ala Ala Ala
100 105 110
Gly Thr Glu Cys Leu Ile Ser Gly Trp Gly Asn Thr Cys Ser Ser Gly
115 120 125
Ser Ser Met Pro Ser Leu Leu Gln Cys Leu Lys Ala Pro Val Leu Ser
130 135 140
Asp Ser Ser Cys Lys Ser Ser Tyr Pro Gly Gln Ile Thr Gly Asn Met
145 150 155 160
Ile Cys Val Gly Phe Leu Glu Gly Gly Lys Asp Ala Cys Gln Gly Asp
165 170 175
Ser Gly Gly Pro Val Val Cys Asn Gly Gln Leu Gln Gly Ile Val Ser
180 185 190
Ser Gly Tyr Gly Cys Ala Gln Lys Asn Lys Pro Gly Val Tyr Thr Lys
195 200 205
Val Cys Asn Tyr Val Asn Trp Ile Gln Gln Thr Ile Ala Ala Asn
210 215 220
<210> 78
<211> 223
<212> PRT
<213> 人工序列
<220>
<223> 猪胰蛋白酶变体78号
<400> 78
Ile Val Gly Gly Tyr Thr Cys Ala Ala Asn Ser Ile Pro Tyr Gln Val
1 5 10 15
Ser Leu Asn Ser Gly Ser His Arg Cys Gly Gly Ser Leu Ile Asn Ser
20 25 30
Gln Trp Val Val Ser Ala Ala His Cys Tyr Lys Ser Arg Ile Gln Val
35 40 45
Arg Leu Gly Glu His Asn Ile His Val Leu Glu Gly Asn Glu Gln Phe
50 55 60
Ile Asn Ala Ala Lys Ile Ile Thr His Pro Asn Phe Asn Pro Asn Thr
65 70 75 80
Leu Asp Asn Asp Ile Met Leu Ile Lys Leu Ser Ser Pro Ala Thr Leu
85 90 95
Asn Ser His Val Ala Thr Val Ser Leu Pro Thr Ser Cys Ala Ala Ala
100 105 110
Gly Thr Glu Cys Leu Ile Ser Gly Trp Gly Asn Thr Cys Ser Ser Gly
115 120 125
Ser Ser Tyr Pro Ser Leu Leu Gln Cys Leu Lys Ala Pro Val Leu Ser
130 135 140
Asp Ser Ser Cys Lys Ser Ser Tyr Pro Gly Gln Ile Thr Gly Asn Met
145 150 155 160
Ile Cys Val Gly Phe Leu Glu Gly Gly Lys Asp Ala Cys Gln Gly Asp
165 170 175
Ser Gly Gly Pro Val Val Cys Asn Gly Gln Leu Gln Gly Ile Val Ser
180 185 190
Ser Gly Tyr Gly Cys Ala Gln Lys Asn Lys Pro Gly Val Tyr Thr Lys
195 200 205
Val Cys Asn Tyr Val Asn Trp Ile Gln Gln Thr Ile Ala Ala Asn
210 215 220
<210> 79
<211> 223
<212> PRT
<213> 人工序列
<220>
<223> 猪胰蛋白酶变体79号
<400> 79
Ile Val Gly Gly Tyr Thr Cys Ala Ala Asn Ser Ile Pro Tyr Gln Val
1 5 10 15
Ser Leu Asn Ser Gly Ser His Arg Cys Gly Gly Ser Leu Ile Asn Ser
20 25 30
Gln Trp Val Val Ser Ala Ala His Cys Tyr Lys Pro Arg Ile Gln Val
35 40 45
Arg Leu Gly Glu His Asn Ile His Val Leu Glu Gly Asn Glu Gln Phe
50 55 60
Ile Asn Ala Ala Lys Ile Ile Thr His Pro Asn Phe Asn Pro Asn Thr
65 70 75 80
Leu Asp Asn Asp Ile Met Leu Ile Lys Leu Ser Ser Pro Ala Thr Leu
85 90 95
Asn Ser His Val Ala Thr Val Ser Leu Pro Arg Ser Cys Ala Ala Ala
100 105 110
Gly Thr Glu Cys Leu Ile Ser Gly Trp Gly Asn Thr Ala Ser Ser Gly
115 120 125
Ser Ser Met Pro Ser Leu Leu Gln Cys Leu Lys Ala Pro Val Leu Ser
130 135 140
Asp Ser Ser Cys Lys Ser Ser Tyr Pro Gly Gln Ile Thr Gly Asn Met
145 150 155 160
Ile Cys Val Gly Phe Leu Glu Gly Gly Lys Asp Ala Cys Gln Gly Asp
165 170 175
Ser Gly Gly Pro Val Val Cys Asn Gly Gln Leu Gln Gly Ile Val Ser
180 185 190
Ser Gly Tyr Gly Cys Ala Gln Lys Asn Lys Pro Gly Val Tyr Thr Lys
195 200 205
Val Cys Asn Tyr Val Asn Trp Ile Gln Gln Thr Ile Ala Ala Asn
210 215 220
<210> 80
<211> 223
<212> PRT
<213> 人工序列
<220>
<223> 猪胰蛋白酶变体80号
<400> 80
Ile Val Gly Gly Tyr Thr Cys Ala Ala Asn Ser Ile Pro Tyr Gln Val
1 5 10 15
Ser Leu Asn Ser Gly Ser His Arg Cys Gly Gly Ser Leu Ile Asn Ser
20 25 30
Gln Trp Val Val Ser Ala Ala His Cys Tyr Lys Ser Arg Ile Gln Val
35 40 45
Arg Leu Gly Glu His Asn Ile His Val Leu Glu Gly Asn Glu Gln Phe
50 55 60
Ile Asn Ala Ala Lys Ile Ile Thr His Pro Asn Phe Asn Pro Asn Thr
65 70 75 80
Leu Asp Asn Asp Ile Met Leu Ile Lys Leu Ser Ser Pro Ala Thr Leu
85 90 95
Asn Ser His Val Ala Thr Val Ser Leu Pro Thr Ser Cys Ala Ala Ala
100 105 110
Gly Thr Glu Cys Leu Ile Ser Gly Trp Gly Asn Thr Ser Ser Ser Gly
115 120 125
Ser Ser Met Pro Ser Leu Leu Gln Cys Leu Lys Ala Pro Val Leu Ser
130 135 140
Asp Ser Ser Cys Lys Ser Ser Tyr Pro Gly Gln Ile Thr Gly Asn Met
145 150 155 160
Ile Cys Val Gly Phe Leu Glu Gly Gly Lys Asp Ala Cys Gln Gly Asp
165 170 175
Ser Gly Gly Pro Val Val Cys Asn Gly Gln Leu Gln Gly Ile Val Ser
180 185 190
Ser Gly Tyr Gly Cys Ala Gln Lys Asn Lys Pro Gly Val Tyr Thr Lys
195 200 205
Val Cys Asn Tyr Val Asn Trp Ile Gln Gln Thr Ile Ala Ala Asn
210 215 220
<210> 81
<211> 223
<212> PRT
<213> 人工序列
<220>
<223> 猪胰蛋白酶变体81号
<400> 81
Ile Val Gly Gly Tyr Thr Cys Ala Ala Asn Ser Ile Pro Tyr Gln Val
1 5 10 15
Ser Leu Asn Ser Gly Ser His Arg Cys Gly Gly Ser Leu Ile Asn Ser
20 25 30
Gln Trp Val Val Ser Ala Ala His Cys Tyr Lys Ser Arg Ile Gln Val
35 40 45
Arg Leu Gly Glu His Asn Ile His Val Leu Glu Gly Asn Glu Gln Phe
50 55 60
Ile Asn Ala Ala Lys Ile Ile Thr His Pro Asn Phe Asn Pro Asn Thr
65 70 75 80
Leu Asp Asn Asp Ile Met Leu Ile Lys Leu Ser Ser Pro Ala Thr Leu
85 90 95
Asn Ser His Val Ala Thr Val Ser Leu Pro Thr Ser Cys Ala Ala Ala
100 105 110
Gly Thr Glu Cys Leu Ile Ser Gly Trp Gly Asn Thr Ala Ser Ser Gly
115 120 125
Ser Ser Met Pro Ser Leu Leu Gln Cys Leu Lys Ala Pro Val Leu Ser
130 135 140
Asp Ser Ser Cys Lys Ser Ser Tyr Pro Gly Gln Ile Thr Gly Asn Met
145 150 155 160
Ile Cys Val Gly Phe Leu Glu Gly Gly Lys Asp Ala Cys Gln Gly Asp
165 170 175
Ser Gly Gly Pro Val Val Cys Asn Gly Gln Leu Gln Gly Ile Val Ser
180 185 190
Ser Gly Tyr Gly Cys Ala Gln Lys Asn Lys Pro Gly Val Tyr Thr Lys
195 200 205
Val Cys Asn Tyr Val Asn Trp Ile Gln Gln Thr Ile Ala Ala Asn
210 215 220
<210> 82
<211> 223
<212> PRT
<213> 人工序列
<220>
<223> 猪胰蛋白酶变体82号
<400> 82
Ile Val Gly Gly Tyr Thr Cys Ala Ala Asn Ser Ile Pro Tyr Gln Val
1 5 10 15
Ser Leu Asn Ser Gly Ser His Arg Cys Gly Gly Ser Leu Ile Asn Ser
20 25 30
Gln Trp Val Val Ser Ala Ala His Cys Tyr Lys Pro Arg Ile Gln Val
35 40 45
Arg Leu Gly Glu His Asn Ile His Val Leu Glu Gly Asn Glu Gln Phe
50 55 60
Ile Asn Ala Ala Lys Ile Ile Thr His Pro Asn Phe Asn Pro Asn Thr
65 70 75 80
Leu Asp Asn Asp Ile Met Leu Ile Lys Leu Ser Ser Pro Ala Thr Leu
85 90 95
Asn Ser His Val Ala Thr Val Ser Leu Pro Arg Ser Cys Ala Ala Ala
100 105 110
Gly Thr Glu Cys Leu Ile Ser Gly Trp Gly Asn Thr Cys Ser Ser Gly
115 120 125
Ser Ser Met Pro Ser Leu Leu Gln Cys Leu Lys Ala Pro Val Leu Ser
130 135 140
Asp Ser Ser Cys Lys Ser Ser Tyr Pro Gly Gln Ile Thr Gly Asn Met
145 150 155 160
Ile Cys Val Gly Phe Leu Glu Gly Gly Lys Asp Ala Cys Gln Gly Asp
165 170 175
Ser Gly Gly Pro Val Val Cys Asn Gly Gln Leu Gln Gly Ile Val Ser
180 185 190
Ser Gly Tyr Gly Cys Ala Gln Lys Asn Lys Pro Gly Val Tyr Thr Lys
195 200 205
Val Cys Asn Tyr Val Asn Trp Ile Gln Gln Thr Ile Ala Ala Asn
210 215 220
<210> 83
<211> 223
<212> PRT
<213> 人工序列
<220>
<223> 猪胰蛋白酶变体83号
<400> 83
Ile Val Gly Gly Tyr Thr Cys Ala Ala Asn Ser Ile Pro Tyr Gln Val
1 5 10 15
Ser Leu Asn Ser Gly Ser His Arg Cys Gly Gly Ser Leu Ile Asn Ser
20 25 30
Gln Trp Val Val Ser Ala Ala His Cys Tyr Lys Pro Arg Ile Gln Val
35 40 45
Arg Leu Gly Glu His Asn Ile His Val Leu Glu Gly Asn Glu Gln Phe
50 55 60
Ile Asn Ala Ala Lys Ile Ile Thr His Pro Asn Phe Asn Pro Asn Thr
65 70 75 80
Leu Asp Asn Asp Ile Met Leu Ile Lys Leu Ser Ser Pro Ala Thr Leu
85 90 95
Asn Ser Asn Val Ala Thr Val Ser Leu Pro Thr Ser Cys Ala Ala Ala
100 105 110
Gly Thr Glu Cys Leu Ile Ser Gly Trp Gly Asn Thr Ser Ser Ser Gly
115 120 125
Ser Ser Met Pro Ser Leu Leu Gln Cys Leu Lys Ala Pro Val Leu Ser
130 135 140
Asp Ser Ser Cys Lys Ser Ser Tyr Pro Gly Gln Ile Thr Gly Asn Met
145 150 155 160
Ile Cys Val Gly Phe Leu Glu Gly Gly Lys Asp Ala Cys Gln Gly Asp
165 170 175
Ser Gly Gly Pro Val Val Cys Asn Gly Gln Leu Gln Gly Ile Val Ser
180 185 190
Ser Gly Tyr Gly Cys Ala Gln Lys Asn Lys Pro Gly Val Tyr Thr Lys
195 200 205
Val Cys Asn Tyr Val Asn Trp Ile Gln Gln Thr Ile Ala Ala Asn
210 215 220
<210> 84
<211> 223
<212> PRT
<213> 人工序列
<220>
<223> 猪胰蛋白酶变体84号
<400> 84
Ile Val Gly Gly Tyr Thr Cys Ala Ala Asn Ser Ile Pro Tyr Gln Val
1 5 10 15
Ser Leu Asn Ser Gly Ser His Arg Cys Gly Gly Ser Leu Ile Asn Ser
20 25 30
Gln Trp Val Val Ser Ala Ala His Cys Tyr Lys Pro Arg Ile Gln Val
35 40 45
Arg Leu Gly Glu His Asn Ile His Val Leu Glu Gly Asn Glu Gln Phe
50 55 60
Ile Asn Ala Ala Lys Ile Ile Thr His Pro Asn Phe Asn Pro Asn Thr
65 70 75 80
Leu Asp Asn Asp Ile Met Leu Ile Lys Leu Ser Ser Pro Ala Thr Leu
85 90 95
Asn Ser Asn Val Ala Thr Val Ser Leu Pro Thr Ser Cys Ala Ala Ala
100 105 110
Gly Thr Glu Cys Leu Ile Ser Gly Trp Gly Asn Thr Ser Ser Ser Gly
115 120 125
Ser Ser Met Pro Ser Leu Leu Gln Cys Leu Lys Ala Pro Val Leu Ser
130 135 140
Asp Ser Ser Cys Lys Ser Ser Tyr Pro Gly Gln Ile Thr Gly Asn Met
145 150 155 160
Ile Cys Val Gly Phe Leu Glu Gly Gly Lys Asp Ser Cys Gln Gly Asp
165 170 175
Ser Gly Gly Pro Val Val Cys Asn Gly Gln Leu Gln Gly Ile Val Ser
180 185 190
Ser Gly Tyr Gly Cys Ala Gln Lys Asn Lys Pro Gly Val Tyr Thr Lys
195 200 205
Val Cys Asn Tyr Val Asn Trp Ile Gln Gln Thr Ile Ala Ala Asn
210 215 220
<210> 85
<211> 223
<212> PRT
<213> 人工序列
<220>
<223> 猪胰蛋白酶变体85号
<400> 85
Ile Val Gly Gly Tyr Thr Cys Ala Ala Asn Ser Ile Pro Tyr Gln Val
1 5 10 15
Ser Leu Asn Ser Gly Ser His Arg Cys Gly Gly Ser Leu Ile Asn Ser
20 25 30
Gln Trp Val Val Ser Ala Ala His Cys Tyr Lys Pro Arg Ile Gln Val
35 40 45
Arg Leu Gly Glu His Asn Ile His Val Leu Glu Gly Asn Glu Gln Phe
50 55 60
Ile Asn Ala Ala Lys Ile Ile Thr His Pro Asn Phe Asn Pro Asn Thr
65 70 75 80
Leu Asp Asn Asp Ile Met Leu Ile Lys Leu Ser Ser Pro Ala Thr Leu
85 90 95
Asn Ser His Val Ala Thr Val Ser Leu Pro Thr Ser Cys Ala Ala Ala
100 105 110
Gly Thr Glu Cys Leu Ile Ser Gly Trp Gly Asn Thr Cys Ser Ser Gly
115 120 125
Ser Ser Met Pro Ser Leu Leu Gln Cys Leu Lys Ala Pro Val Leu Ser
130 135 140
Asp Ser Ser Cys Lys Ser Ser Tyr Pro Gly Gln Ile Thr Gly Asn Met
145 150 155 160
Ile Cys Val Gly Phe Leu Glu Gly Gly Lys Asp Ala Cys Gln Gly Asp
165 170 175
Ser Gly Gly Pro Val Val Cys Asn Gly Gln Leu Gln Gly Ile Val Ser
180 185 190
Ser Gly Tyr Gly Cys Ala Gln Lys Asn Lys Pro Gly Val Tyr Thr Lys
195 200 205
Val Cys Asn Tyr Val Asn Trp Ile Gln Gln Thr Ile Ala Ala Asn
210 215 220
<210> 86
<211> 223
<212> PRT
<213> 人工序列
<220>
<223> 猪胰蛋白酶变体86号
<400> 86
Ile Val Gly Gly Tyr Thr Cys Ala Ala Asn Ser Ile Pro Tyr Gln Val
1 5 10 15
Ser Leu Asn Ser Gly Ser His Arg Cys Gly Gly Ser Leu Ile Asn Ser
20 25 30
Gln Trp Val Val Ser Ala Ala His Cys Tyr Lys Pro Arg Ile Gln Val
35 40 45
Arg Leu Gly Glu His Asn Ile Asp Val Leu Glu Gly Asn Glu Gln Phe
50 55 60
Ile Asn Ala Ala Lys Ile Ile Thr His Pro Asn Phe Asn Pro Asn Thr
65 70 75 80
Leu Asp Asn Asp Ile Met Leu Ile Lys Leu Ser Ser Pro Ala Thr Leu
85 90 95
Asn Ser Ala Val Ala Thr Val Ser Leu Pro Thr Ser Cys Ala Ala Ala
100 105 110
Gly Thr Glu Cys Leu Ile Ser Gly Trp Gly Asn Thr Ser Ser Ser Gly
115 120 125
Ser Ser Met Pro Ser Leu Leu Gln Cys Leu Lys Ala Pro Val Leu Ser
130 135 140
Asp Ser Ser Cys Lys Ser Ser Tyr Pro Gly Gln Ile Thr Gly Asn Met
145 150 155 160
Ile Cys Val Gly Phe Leu Glu Gly Gly Lys Asp Ala Cys Gln Gly Asp
165 170 175
Ser Gly Gly Pro Val Val Cys Asn Gly Gln Leu Gln Gly Ile Val Ser
180 185 190
Ser Gly Tyr Gly Cys Ala Gln Lys Asn Lys Pro Gly Val Tyr Thr Lys
195 200 205
Val Cys Asn Tyr Val Asn Trp Ile Gln Gln Thr Ile Ala Ala Asn
210 215 220
<210> 87
<211> 223
<212> PRT
<213> 人工序列
<220>
<223> 猪胰蛋白酶变体87号
<400> 87
Ile Val Gly Gly Tyr Thr Cys Ala Ala Asn Ser Ile Pro Tyr Gln Val
1 5 10 15
Ser Leu Asn Ser Gly Ser His Arg Cys Gly Gly Ser Leu Ile Asn Ser
20 25 30
Gln Trp Val Val Ser Ala Ala His Cys Tyr Lys Pro Arg Ile Gln Val
35 40 45
Arg Leu Gly Glu His Asn Ile His Val Leu Glu Gly Asn Glu Gln Phe
50 55 60
Ile Asn Ala Ala Lys Ile Ile Thr His Pro Asn Phe Asn Pro Asn Thr
65 70 75 80
Leu Asp Asn Asp Ile Met Leu Ile Lys Leu Ser Ser Pro Ala Thr Leu
85 90 95
Asn Ser His Val Ala Thr Val Ser Leu Pro Thr Ser Cys Ala Ala Ala
100 105 110
Gly Thr Glu Cys Leu Ile Ser Gly Trp Gly Asn Thr Ser Ser Ser Gly
115 120 125
Ser Ser Met Pro Ser Leu Leu Gln Cys Leu Lys Ala Pro Val Leu Ser
130 135 140
Asp Ser Ser Cys Lys Ser Ser Tyr Pro Gly Gln Ile Thr Gly Asn Met
145 150 155 160
Ile Cys Val Gly Phe Leu Glu Gly Gly Lys Asp Ala Cys Gln Gly Asp
165 170 175
Ser Gly Gly Pro Val Val Cys Asn Gly Gln Leu Gln Gly Ile Val Ser
180 185 190
Ser Gly Tyr Gly Cys Ala Gln Lys Asn Lys Pro Gly Val Tyr Thr Lys
195 200 205
Val Cys Asn Tyr Val Asn Trp Ile Gln Gln Thr Ile Ala Ala Asn
210 215 220
<210> 88
<211> 223
<212> PRT
<213> 人工序列
<220>
<223> 猪胰蛋白酶变体88号
<400> 88
Ile Val Gly Gly Tyr Thr Cys Ala Ala Asn Ser Ile Pro Tyr Gln Val
1 5 10 15
Ser Leu Asn Ser Gly Ser His Arg Cys Gly Gly Ser Leu Ile Asn Ser
20 25 30
Gln Trp Val Val Ser Ala Ala His Cys Tyr Lys Pro Arg Ile Gln Val
35 40 45
Arg Leu Gly Glu His Asn Ile His Val Leu Glu Gly Asn Glu Gln Phe
50 55 60
Ile Asn Ala Ala Lys Ile Ile Thr His Pro Asn Phe Asn Pro Asn Thr
65 70 75 80
Leu Asp Asn Asp Ile Met Leu Ile Lys Leu Ser Ser Pro Ala Thr Leu
85 90 95
Asn Ser His Val Ala Thr Val Ser Leu Pro Thr Ser Cys Ala Ala Ala
100 105 110
Gly Thr Glu Cys Leu Ile Ser Gly Trp Gly Asn Thr Ser Ser Ser Gly
115 120 125
Ser Ser Met Pro Ser Leu Leu Gln Cys Leu Lys Ala Pro Val Leu Ser
130 135 140
Asp Ser Ser Cys Lys Ser Ser Tyr Pro Gly Gln Ile Thr Gly Asn Met
145 150 155 160
Ile Cys Val Gly Phe Leu Glu Gly Gly Lys Asp Ser Cys Gln Gly Asp
165 170 175
Ser Gly Gly Pro Val Val Cys Asn Gly Gln Leu Gln Gly Ile Val Ser
180 185 190
Ser Gly Tyr Gly Cys Ala Gln Lys Asn Lys Pro Gly Val Tyr Thr Lys
195 200 205
Val Cys Asn Tyr Val Asn Trp Ile Gln Gln Thr Ile Ala Ala Asn
210 215 220
<210> 89
<211> 223
<212> PRT
<213> 人工序列
<220>
<223> 猪胰蛋白酶变体89号
<400> 89
Ile Val Gly Gly Tyr Thr Cys Ala Ala Asn Ser Ile Pro Tyr Gln Val
1 5 10 15
Ser Leu Asn Ser Gly Ser His Arg Cys Gly Gly Ser Leu Ile Asn Ser
20 25 30
Gln Trp Val Val Ser Ala Ala His Cys Tyr Lys Pro Arg Ile Gln Val
35 40 45
Arg Leu Gly Glu His Asn Ile His Val Leu Glu Gly Asn Glu Gln Phe
50 55 60
Ile Asn Ala Ala Lys Ile Ile Thr His Pro Asn Phe Asn Pro Asn Thr
65 70 75 80
Leu Asp Asn Asp Ile Met Leu Ile Lys Leu Ser Ser Pro Ala Thr Leu
85 90 95
Asn Ser His Val Ala Thr Val Ser Leu Pro Thr Ser Cys Ala Ala Ala
100 105 110
Gly Thr Glu Cys Leu Ile Ser Gly Trp Gly Asn Thr Ala Ser Ser Gly
115 120 125
Ser Ser Met Pro Ser Leu Leu Gln Cys Leu Lys Ala Pro Val Leu Ser
130 135 140
Asp Ser Ser Cys Lys Ser Ser Tyr Pro Gly Gln Ile Thr Gly Asn Met
145 150 155 160
Ile Cys Val Gly Phe Leu Glu Gly Gly Lys Asp Ala Cys Gln Gly Asp
165 170 175
Ser Gly Gly Pro Val Val Cys Asn Gly Gln Leu Gln Gly Ile Val Ser
180 185 190
Ser Gly Tyr Gly Cys Ala Gln Lys Asn Lys Pro Gly Val Tyr Thr Lys
195 200 205
Val Cys Asn Tyr Val Asn Trp Ile Gln Gln Thr Ile Ala Ala Asn
210 215 220
<210> 90
<211> 223
<212> PRT
<213> 人工序列
<220>
<223> 猪胰蛋白酶变体90号
<400> 90
Ile Val Gly Gly Tyr Thr Cys Ala Ala Asn Ser Ile Pro Tyr Gln Val
1 5 10 15
Ser Leu Asn Ser Gly Ser His Arg Cys Gly Gly Ser Leu Ile Asn Ser
20 25 30
Gln Trp Val Val Ser Ala Ala His Cys Tyr Lys Pro Arg Ile Gln Val
35 40 45
Arg Leu Gly Glu His Asn Ile His Val Leu Glu Gly Asn Glu Gln Phe
50 55 60
Ile Asn Ala Ala Lys Ile Ile Thr His Pro Asn Phe Asn Pro Asn Thr
65 70 75 80
Leu Asp Asn Asp Ile Met Leu Ile Lys Leu Ser Ser Pro Ala Thr Leu
85 90 95
Asn Ser His Val Ala Thr Val Ser Leu Pro Thr Ser Cys Ala Ala Ala
100 105 110
Gly Thr Glu Cys Leu Ile Ser Gly Trp Gly Asn Thr Ala Ser Ser Gly
115 120 125
Ser Ser Met Pro Ser Leu Leu Gln Cys Leu Lys Ala Pro Val Leu Ser
130 135 140
Asp Ser Ser Cys Lys Ser Ser Tyr Pro Gly Gln Ile Thr Gly Asn Met
145 150 155 160
Ile Cys Val Gly Phe Leu Glu Gly Gly Lys Asp Ser Cys Gln Gly Asp
165 170 175
Ser Gly Gly Pro Val Val Cys Asn Gly Gln Leu Gln Gly Ile Val Ser
180 185 190
Ser Gly Tyr Gly Cys Ala Gln Lys Asn Lys Pro Gly Val Tyr Thr Lys
195 200 205
Val Cys Asn Tyr Val Asn Trp Ile Gln Gln Thr Ile Ala Ala Asn
210 215 220
<210> 91
<211> 223
<212> PRT
<213> 人工序列
<220>
<223> 猪胰蛋白酶变体91号
<400> 91
Ile Val Gly Gly Tyr Thr Cys Ala Ala Asn Ser Ile Pro Tyr Gln Val
1 5 10 15
Ser Leu Asn Ser Gly Ser His Arg Cys Gly Gly Ser Leu Ile Asn Ser
20 25 30
Gln Trp Val Val Ser Ala Ala His Cys Tyr Lys Pro Arg Ile Gln Val
35 40 45
Arg Leu Gly Glu His Asn Ile His Val Leu Glu Gly Asn Glu Gln Phe
50 55 60
Ile Asn Ala Ala Lys Ile Ile Thr His Pro Asn Phe Asn Pro Asn Thr
65 70 75 80
Leu Asp Asn Asp Ile Met Leu Ile Lys Leu Ser Ser Pro Ala Thr Leu
85 90 95
Asn Ser Ala Val Ala Thr Val Ser Leu Pro Thr Ser Cys Ala Ala Ala
100 105 110
Gly Thr Glu Cys Leu Ile Ser Gly Trp Gly Asn Thr Ser Ser Ser Gly
115 120 125
Ser Ser Met Pro Ser Leu Leu Gln Cys Leu Lys Ala Pro Val Leu Ser
130 135 140
Asp Ser Ser Cys Lys Ser Ser Tyr Pro Gly Gln Ile Thr Gly Asn Met
145 150 155 160
Ile Cys Val Gly Phe Leu Glu Gly Gly Lys Asp Ala Cys Gln Gly Asp
165 170 175
Ser Gly Gly Pro Val Val Cys Asn Gly Gln Leu Gln Gly Ile Val Ser
180 185 190
Ser Gly Tyr Gly Cys Ala Gln Lys Asn Lys Pro Gly Val Tyr Thr Lys
195 200 205
Val Cys Asn Tyr Val Asn Trp Ile Gln Gln Thr Ile Ala Ala Asn
210 215 220
<210> 92
<211> 223
<212> PRT
<213> 人工序列
<220>
<223> 猪胰蛋白酶变体92号
<400> 92
Ile Val Gly Gly Tyr Thr Cys Ala Ala Asn Ser Ile Pro Tyr Gln Val
1 5 10 15
Ser Leu Asn Ser Gly Ser His Arg Cys Gly Gly Ser Leu Ile Asn Ser
20 25 30
Gln Trp Val Val Ser Ala Ala His Cys Tyr Lys Pro Arg Ile Gln Val
35 40 45
Arg Leu Gly Glu His Asn Ile His Val Leu Glu Gly Asn Glu Gln Phe
50 55 60
Ile Asn Ala Ala Lys Ile Ile Thr His Pro Asn Phe Asn Pro Asn Thr
65 70 75 80
Leu Asp Asn Asp Ile Met Leu Ile Lys Leu Ser Ser Pro Ala Thr Leu
85 90 95
Asn Ser Ala Val Ala Thr Val Ser Leu Pro Thr Ser Cys Ala Ala Ala
100 105 110
Gly Thr Glu Cys Leu Ile Ser Gly Trp Gly Asn Thr Cys Ser Ser Gly
115 120 125
Ser Ser Met Pro Ser Leu Leu Gln Cys Leu Lys Ala Pro Val Leu Ser
130 135 140
Asp Ser Ser Cys Lys Ser Ser Tyr Pro Gly Gln Ile Thr Gly Asn Met
145 150 155 160
Ile Cys Val Gly Phe Leu Glu Gly Gly Lys Asp Ala Cys Gln Gly Asp
165 170 175
Ser Gly Gly Pro Val Val Cys Asn Gly Gln Leu Gln Gly Ile Val Ser
180 185 190
Ser Gly Tyr Gly Cys Ala Gln Lys Asn Lys Pro Gly Val Tyr Thr Lys
195 200 205
Val Cys Asn Tyr Val Asn Trp Ile Gln Gln Thr Ile Ala Ala Asn
210 215 220
<210> 93
<211> 223
<212> PRT
<213> 人工序列
<220>
<223> 猪胰蛋白酶变体93号
<400> 93
Ile Val Gly Gly Tyr Thr Cys Ala Ala Asn Ser Ile Pro Tyr Gln Val
1 5 10 15
Ser Leu Asn Ser Gly Ser His Arg Cys Gly Gly Ser Leu Ile Asn Ser
20 25 30
Gln Trp Val Val Ser Ala Ala His Cys Tyr Lys Ser Arg Ile Gln Val
35 40 45
Arg Leu Gly Glu His Asn Ile Asp Val Leu Glu Gly Asn Glu Gln Phe
50 55 60
Ile Asn Ala Ala Lys Ile Ile Thr His Pro Asn Phe Asn Pro Asn Thr
65 70 75 80
Leu Asp Asn Asp Ile Met Leu Ile Lys Leu Ser Ser Pro Ala Thr Leu
85 90 95
Asn Ser Asn Val Ala Thr Val Ser Leu Pro Thr Ser Cys Ala Ala Ala
100 105 110
Gly Thr Glu Cys Leu Ile Ser Gly Trp Gly Asn Thr Cys Ser Ser Gly
115 120 125
Ser Ser Met Pro Ser Leu Leu Gln Cys Leu Lys Ala Pro Val Leu Ser
130 135 140
Asp Ser Ser Cys Lys Ser Ser Tyr Pro Gly Gln Ile Thr Gly Asn Met
145 150 155 160
Ile Cys Val Gly Phe Leu Glu Gly Gly Ala Asp Ala Cys Gln Gly Asp
165 170 175
Ser Gly Gly Pro Val Val Cys Asn Gly Gln Leu Gln Gly Ile Val Ser
180 185 190
Ser Gly Tyr Gly Cys Ala Gln Lys Asn Lys Pro Gly Val Tyr Thr Lys
195 200 205
Val Cys Asn Tyr Val Asn Trp Ile Gln Gln Thr Ile Ala Ala Asn
210 215 220
<210> 94
<211> 223
<212> PRT
<213> 人工序列
<220>
<223> 猪胰蛋白酶变体94号
<400> 94
Ile Val Gly Gly Tyr Thr Cys Ala Ala Asn Ser Ile Pro Tyr Gln Val
1 5 10 15
Ser Leu Asn Ser Gly Ser His Arg Cys Gly Gly Ser Leu Ile Asn Ser
20 25 30
Gln Trp Val Val Ser Ala Ala His Cys Tyr Lys Pro Arg Ile Gln Val
35 40 45
Arg Leu Gly Glu His Asn Ile His Val Leu Glu Gly Asn Glu Gln Phe
50 55 60
Ile Asn Ala Ala Lys Ile Ile Thr His Pro Asn Phe Asn Gly Asn Thr
65 70 75 80
Leu Asp Asn Asp Ile Met Leu Ile Lys Leu Ser Ser Pro Ala Thr Leu
85 90 95
Asn Ser Asn Val Ala Thr Val Ser Leu Pro Arg Ser Cys Ala Ala Ala
100 105 110
Gly Thr Glu Cys Leu Ile Ser Gly Trp Gly Asn Thr Cys Ser Ser Gly
115 120 125
Ser Ser Met Pro Ser Leu Leu Gln Cys Leu Lys Ala Pro Val Leu Ser
130 135 140
Asp Ser Ser Cys Lys Ser Ser Tyr Pro Gly Gln Ile Thr Gly Asn Met
145 150 155 160
Ile Cys Val Gly Phe Leu Glu Gly Gly Ala Asp Ala Cys Gln Gly Asp
165 170 175
Ser Gly Gly Pro Val Val Cys Asn Gly Gln Leu Gln Gly Ile Val Ser
180 185 190
Ser Gly Tyr Gly Cys Ala Gln Lys Asn Lys Pro Gly Val Tyr Thr Lys
195 200 205
Val Cys Asn Tyr Val Asn Trp Ile Gln Gln Thr Ile Ala Ala Asn
210 215 220
<210> 95
<211> 223
<212> PRT
<213> 人工序列
<220>
<223> 猪胰蛋白酶变体95号
<400> 95
Ile Val Gly Gly Tyr Thr Cys Ala Ala Asn Ser Ile Pro Tyr Gln Val
1 5 10 15
Ser Leu Asn Ser Gly Ser His Arg Cys Gly Gly Ser Leu Ile Asn Ser
20 25 30
Gln Trp Val Val Ser Ala Ala His Cys Tyr Lys Pro Arg Ile Gln Val
35 40 45
Arg Leu Gly Glu His Asn Ile His Val Leu Glu Gly Asn Glu Gln Phe
50 55 60
Ile Asn Ala Ala Lys Ile Ile Thr His Pro Asn Phe Asn Pro Asn Thr
65 70 75 80
Leu Asp Asn Asp Ile Met Leu Ile Lys Leu Ser Ser Pro Ala Thr Leu
85 90 95
Asn Ser His Val Ala Thr Val Ser Leu Pro Arg Ser Cys Ala Ala Ala
100 105 110
Gly Thr Glu Cys Leu Ile Ser Gly Trp Gly Asn Thr Ser Ser Ser Gly
115 120 125
Ser Ser Met Pro Ser Leu Leu Gln Cys Leu Lys Ala Pro Val Leu Ser
130 135 140
Asp Ser Ser Cys Lys Ser Ser Tyr Pro Gly Gln Ile Thr Gly Asn Met
145 150 155 160
Ile Cys Val Gly Phe Leu Glu Gly Gly Ala Asp Ala Cys Gln Gly Asp
165 170 175
Ser Gly Gly Pro Val Val Cys Asn Gly Gln Leu Gln Gly Ile Val Ser
180 185 190
Ser Gly Tyr Gly Cys Ala Gln Lys Asn Lys Pro Gly Val Tyr Thr Lys
195 200 205
Val Cys Asn Tyr Val Asn Trp Ile Gln Gln Thr Ile Ala Ala Asn
210 215 220
<210> 96
<211> 223
<212> PRT
<213> 人工序列
<220>
<223> 猪胰蛋白酶变体96号
<400> 96
Ile Val Gly Gly Tyr Thr Cys Ala Ala Asn Ser Ile Pro Tyr Gln Val
1 5 10 15
Ser Leu Asn Ser Gly Ser His Arg Cys Gly Gly Ser Leu Ile Asn Ser
20 25 30
Gln Trp Val Val Ser Ala Ala His Cys Tyr Lys Pro Arg Ile Gln Val
35 40 45
Arg Leu Gly Glu His Asn Ile Asp Val Leu Glu Gly Asn Glu Gln Phe
50 55 60
Ile Asn Ala Ala Lys Ile Ile Thr His Pro Asn Phe Asn Pro Asn Thr
65 70 75 80
Leu Asp Asn Asp Ile Met Leu Ile Lys Leu Ser Ser Pro Ala Thr Leu
85 90 95
Asn Ser His Val Ala Thr Val Ser Leu Pro Arg Ser Cys Ala Ala Ala
100 105 110
Gly Thr Glu Cys Leu Ile Ser Gly Trp Gly Asn Thr Ser Ser Ser Gly
115 120 125
Ser Ser Met Pro Ser Leu Leu Gln Cys Leu Lys Ala Pro Val Leu Ser
130 135 140
Asp Ser Ser Cys Lys Ser Ser Tyr Pro Gly Gln Ile Thr Gly Asn Met
145 150 155 160
Ile Cys Val Gly Phe Leu Glu Gly Gly Ala Asp Ala Cys Gln Gly Asp
165 170 175
Ser Gly Gly Pro Val Val Cys Asn Gly Gln Leu Gln Gly Ile Val Ser
180 185 190
Ser Gly Tyr Gly Cys Ala Gln Lys Asn Lys Pro Gly Val Tyr Thr Lys
195 200 205
Val Cys Asn Tyr Val Asn Trp Ile Gln Gln Thr Ile Ala Ala Asn
210 215 220
<210> 97
<211> 223
<212> PRT
<213> 人工序列
<220>
<223> 猪胰蛋白酶变体97号
<400> 97
Ile Val Gly Gly Tyr Thr Cys Ala Ala Asn Ser Ile Pro Tyr Gln Val
1 5 10 15
Ser Leu Asn Ser Gly Ser His Arg Cys Gly Gly Ser Leu Ile Asn Ser
20 25 30
Gln Trp Val Val Ser Ala Ala His Cys Tyr Lys Pro Arg Ile Gln Val
35 40 45
Arg Leu Gly Glu His Asn Ile His Val Leu Glu Gly Asn Glu Gln Phe
50 55 60
Ile Asn Ala Ala Lys Ile Ile Thr His Pro Asn Phe Asn Pro Asn Thr
65 70 75 80
Leu Asp Asn Asp Ile Met Leu Ile Lys Leu Ser Ser Pro Ala Thr Leu
85 90 95
Asn Ser Ala Val Ala Thr Val Ser Leu Pro Thr Ser Cys Ala Ala Ala
100 105 110
Gly Thr Glu Cys Leu Ile Ser Gly Trp Gly Asn Thr Ala Ser Ser Gly
115 120 125
Ser Ser Met Pro Ser Leu Leu Gln Cys Leu Lys Ala Pro Val Leu Ser
130 135 140
Asp Ser Ser Cys Lys Ser Ser Tyr Pro Gly Gln Ile Thr Gly Asn Met
145 150 155 160
Ile Cys Val Gly Phe Leu Glu Gly Gly Ala Asp Ala Cys Gln Gly Asp
165 170 175
Ser Gly Gly Pro Val Val Cys Asn Gly Gln Leu Gln Gly Ile Val Ser
180 185 190
Ser Gly Tyr Gly Cys Ala Gln Lys Asn Lys Pro Gly Val Tyr Thr Lys
195 200 205
Val Cys Asn Tyr Val Asn Trp Ile Gln Gln Thr Ile Ala Ala Asn
210 215 220
<210> 98
<211> 223
<212> PRT
<213> 人工序列
<220>
<223> 猪胰蛋白酶变体98号
<400> 98
Ile Val Gly Gly Tyr Thr Cys Ala Ala Asn Ser Ile Pro Tyr Gln Val
1 5 10 15
Ser Leu Asn Ser Gly Ser His Arg Cys Gly Gly Ser Leu Ile Asn Ser
20 25 30
Gln Trp Val Val Ser Ala Ala His Cys Tyr Lys Pro Arg Ile Gln Val
35 40 45
Arg Leu Gly Glu His Asn Ile His Val Leu Glu Gly Asn Glu Gln Phe
50 55 60
Ile Asn Ala Ala Lys Ile Ile Thr His Pro Asn Phe Asn Pro Asn Thr
65 70 75 80
Leu Asp Asn Asp Ile Met Leu Ile Lys Leu Ser Ser Pro Ala Thr Leu
85 90 95
Asn Ser Ala Val Ala Thr Val Ser Leu Pro Thr Ser Cys Ala Ala Ala
100 105 110
Gly Thr Glu Cys Leu Ile Ser Gly Trp Gly Asn Thr Ala Ser Ser Gly
115 120 125
Ser Ser Met Pro Ser Leu Leu Gln Cys Leu Lys Ala Pro Val Leu Ser
130 135 140
Asp Ser Ser Cys Lys Ser Ser Tyr Pro Gly Gln Ile Thr Gly Asn Met
145 150 155 160
Ile Cys Val Gly Phe Leu Glu Gly Gly Ala Asp Ser Cys Gln Gly Asp
165 170 175
Ser Gly Gly Pro Val Val Cys Asn Gly Gln Leu Gln Gly Ile Val Ser
180 185 190
Ser Gly Tyr Gly Cys Ala Gln Lys Asn Lys Pro Gly Val Tyr Thr Lys
195 200 205
Val Cys Asn Tyr Val Asn Trp Ile Gln Gln Thr Ile Ala Ala Asn
210 215 220
<210> 99
<211> 223
<212> PRT
<213> 人工序列
<220>
<223> 猪胰蛋白酶变体99号
<400> 99
Ile Val Gly Gly Tyr Thr Cys Ala Ala Asn Ser Ile Pro Tyr Gln Val
1 5 10 15
Ser Leu Asn Ser Gly Ser His Arg Cys Gly Gly Ser Leu Ile Asn Ser
20 25 30
Gln Trp Val Val Ser Ala Ala His Cys Tyr Lys Ser Arg Ile Gln Val
35 40 45
Arg Leu Gly Glu His Asn Ile His Val Leu Glu Gly Asn Glu Gln Phe
50 55 60
Ile Asn Ala Ala Lys Ile Ile Thr His Pro Asn Phe Asn Pro Asn Thr
65 70 75 80
Leu Asp Asn Asp Ile Met Leu Ile Lys Leu Ser Ser Pro Ala Thr Leu
85 90 95
Asn Ser Asn Val Ala Thr Val Ser Leu Pro Thr Ser Cys Ala Ala Ala
100 105 110
Gly Thr Glu Cys Leu Ile Ser Gly Trp Gly Asn Thr Ser Ser Ser Gly
115 120 125
Ser Ser Tyr Pro Ser Leu Leu Gln Cys Leu Lys Ala Pro Val Leu Ser
130 135 140
Asp Ser Ser Cys Lys Ser Ser Tyr Pro Gly Gln Ile Thr Gly Asn Met
145 150 155 160
Ile Cys Val Gly Phe Leu Glu Gly Gly Ala Asp Ala Cys Gln Gly Asp
165 170 175
Ser Gly Gly Pro Val Val Cys Asn Gly Gln Leu Gln Gly Ile Val Ser
180 185 190
Ser Gly Tyr Gly Cys Ala Gln Lys Asn Lys Pro Gly Val Tyr Thr Lys
195 200 205
Val Cys Asn Tyr Val Asn Trp Ile Gln Gln Thr Ile Ala Ala Asn
210 215 220
<210> 100
<211> 223
<212> PRT
<213> 人工序列
<220>
<223> 猪胰蛋白酶变体100号
<400> 100
Ile Val Gly Gly Tyr Thr Cys Ala Ala Asn Ser Ile Pro Tyr Gln Val
1 5 10 15
Ser Leu Asn Ser Gly Ser His Arg Cys Gly Gly Ser Leu Ile Asn Ser
20 25 30
Gln Trp Val Val Ser Ala Ala His Cys Tyr Lys Pro Arg Ile Gln Val
35 40 45
Arg Leu Gly Glu His Asn Ile Asp Val Leu Glu Gly Asn Glu Gln Phe
50 55 60
Ile Asn Ala Ala Lys Ile Ile Thr His Pro Asn Phe Asn Pro Asn Thr
65 70 75 80
Leu Asp Asn Asp Ile Met Leu Ile Lys Leu Ser Ser Pro Ala Thr Leu
85 90 95
Asn Ser His Val Ala Thr Val Ser Leu Pro Thr Ser Cys Ala Ala Ala
100 105 110
Gly Thr Glu Cys Leu Ile Ser Gly Trp Gly Asn Thr Cys Ser Ser Gly
115 120 125
Ser Ser Met Pro Ser Leu Leu Gln Cys Leu Lys Ala Pro Val Leu Ser
130 135 140
Asp Ser Ser Cys Lys Ser Ser Tyr Pro Gly Gln Ile Thr Gly Asn Met
145 150 155 160
Ile Cys Val Gly Phe Leu Glu Gly Gly Ala Asp Ala Cys Gln Gly Asp
165 170 175
Ser Gly Gly Pro Val Val Cys Asn Gly Gln Leu Gln Gly Ile Val Ser
180 185 190
Ser Gly Tyr Gly Cys Ala Gln Lys Asn Lys Pro Gly Val Tyr Thr Lys
195 200 205
Val Cys Asn Tyr Val Asn Trp Ile Gln Gln Thr Ile Ala Ala Asn
210 215 220
<210> 101
<211> 223
<212> PRT
<213> 人工序列
<220>
<223> 猪胰蛋白酶变体101号
<400> 101
Ile Val Gly Gly Tyr Thr Cys Ala Ala Asn Ser Ile Pro Tyr Gln Val
1 5 10 15
Ser Leu Asn Ser Gly Ser His Arg Cys Gly Gly Ser Leu Ile Asn Ser
20 25 30
Gln Trp Val Val Ser Ala Ala His Cys Tyr Lys Ser Arg Ile Gln Val
35 40 45
Arg Leu Gly Glu His Asn Ile His Val Leu Glu Gly Asn Glu Gln Phe
50 55 60
Ile Asn Ala Ala Lys Ile Ile Thr His Pro Asn Phe Asn Pro Asn Thr
65 70 75 80
Leu Asp Asn Asp Ile Met Leu Ile Lys Leu Ser Ser Pro Ala Thr Leu
85 90 95
Asn Ser Asn Val Ala Thr Val Ser Leu Pro Thr Ser Cys Ala Ala Ala
100 105 110
Gly Thr Glu Cys Leu Ile Ser Gly Trp Gly Asn Thr Ser Ser Ser Gly
115 120 125
Ser Ser Met Pro Ser Leu Leu Gln Cys Leu Lys Ala Pro Val Leu Ser
130 135 140
Asp Ser Ser Cys Lys Ser Ser Tyr Pro Gly Gln Ile Thr Gly Asn Met
145 150 155 160
Ile Cys Val Gly Phe Leu Glu Gly Gly Ala Asp Ala Cys Gln Gly Asp
165 170 175
Ser Gly Gly Pro Val Val Cys Asn Gly Gln Leu Gln Gly Ile Val Ser
180 185 190
Ser Gly Tyr Gly Cys Ala Gln Lys Asn Lys Pro Gly Val Tyr Thr Lys
195 200 205
Val Cys Asn Tyr Val Asn Trp Ile Gln Gln Thr Ile Ala Ala Asn
210 215 220
<210> 102
<211> 223
<212> PRT
<213> 人工序列
<220>
<223> 猪胰蛋白酶变体102号
<400> 102
Ile Val Gly Gly Tyr Thr Cys Ala Ala Asn Ser Ile Pro Tyr Gln Val
1 5 10 15
Ser Leu Asn Ser Gly Ser His Arg Cys Gly Gly Ser Leu Ile Asn Ser
20 25 30
Gln Trp Val Val Ser Ala Ala His Cys Tyr Lys Pro Arg Ile Gln Val
35 40 45
Arg Leu Gly Glu His Asn Ile His Val Leu Glu Gly Asn Glu Gln Phe
50 55 60
Ile Asn Ala Ala Lys Ile Ile Thr His Pro Asn Phe Asn Pro Asn Thr
65 70 75 80
Leu Asp Asn Asp Ile Met Leu Ile Lys Leu Ser Ser Pro Ala Thr Leu
85 90 95
Asn Ser His Val Ala Thr Val Ser Leu Pro Thr Ser Cys Ala Ala Ala
100 105 110
Gly Thr Glu Cys Leu Ile Ser Gly Trp Gly Asn Thr Ser Ser Ser Gly
115 120 125
Ser Ser Met Pro Ser Leu Leu Gln Cys Leu Lys Ala Pro Val Leu Ser
130 135 140
Asp Ser Ser Cys Lys Ser Ser Tyr Pro Gly Gln Ile Thr Gly Asn Met
145 150 155 160
Ile Cys Val Gly Phe Leu Glu Gly Gly Ala Asp Ala Cys Gln Gly Asp
165 170 175
Ser Gly Gly Pro Val Val Cys Asn Gly Gln Leu Gln Gly Ile Val Ser
180 185 190
Ser Gly Tyr Gly Cys Ala Gln Lys Asn Lys Pro Gly Val Tyr Thr Lys
195 200 205
Val Cys Asn Tyr Val Asn Trp Ile Gln Gln Thr Ile Ala Ala Asn
210 215 220
<210> 103
<211> 223
<212> PRT
<213> 人工序列
<220>
<223> 猪胰蛋白酶变体103号
<400> 103
Ile Val Gly Gly Tyr Thr Cys Ala Ala Asn Ser Ile Pro Tyr Gln Val
1 5 10 15
Ser Leu Asn Ser Gly Ser His Arg Cys Gly Gly Ser Leu Ile Asn Ser
20 25 30
Gln Trp Val Val Ser Ala Ala His Cys Tyr Lys Pro Arg Ile Gln Val
35 40 45
Arg Leu Gly Glu His Asn Ile His Val Leu Glu Gly Asn Glu Gln Phe
50 55 60
Ile Asn Ala Ala Lys Ile Ile Thr His Pro Asn Phe Asn Pro Asn Thr
65 70 75 80
Leu Asp Asn Asp Ile Met Leu Ile Lys Leu Ser Ser Pro Ala Thr Leu
85 90 95
Asn Ser His Val Ala Thr Val Ser Leu Pro Thr Ser Cys Ala Ala Ala
100 105 110
Gly Thr Glu Cys Leu Ile Ser Gly Trp Gly Asn Thr Ser Ser Ser Gly
115 120 125
Ser Ser Met Pro Ser Leu Leu Gln Cys Leu Lys Ala Pro Val Leu Ser
130 135 140
Asp Ser Ser Cys Lys Ser Ser Tyr Pro Gly Gln Ile Thr Gly Asn Met
145 150 155 160
Ile Cys Val Gly Phe Leu Glu Gly Gly Ala Asp Ser Cys Gln Gly Asp
165 170 175
Ser Gly Gly Pro Val Val Cys Asn Gly Gln Leu Gln Gly Ile Val Ser
180 185 190
Ser Gly Tyr Gly Cys Ala Gln Lys Asn Lys Pro Gly Val Tyr Thr Lys
195 200 205
Val Cys Asn Tyr Val Asn Trp Ile Gln Gln Thr Ile Ala Ala Asn
210 215 220
<210> 104
<211> 223
<212> PRT
<213> 人工序列
<220>
<223> 猪胰蛋白酶变体104号
<400> 104
Ile Val Gly Gly Tyr Thr Cys Ala Ala Asn Ser Ile Pro Tyr Gln Val
1 5 10 15
Ser Leu Asn Ser Gly Ser His Arg Cys Gly Gly Ser Leu Ile Asn Ser
20 25 30
Gln Trp Val Val Ser Ala Ala His Cys Tyr Lys Ser Arg Ile Gln Val
35 40 45
Arg Leu Gly Glu His Asn Ile His Val Leu Glu Gly Asn Glu Gln Phe
50 55 60
Ile Asn Ala Ala Lys Ile Ile Thr His Pro Asn Phe Asn Pro Asn Thr
65 70 75 80
Leu Asp Asn Asp Ile Met Leu Ile Lys Leu Ser Ser Pro Ala Thr Leu
85 90 95
Asn Ser Asn Val Ala Thr Val Ser Leu Pro Thr Ser Cys Ala Ala Ala
100 105 110
Gly Thr Glu Cys Leu Ile Ser Gly Trp Gly Asn Thr Cys Ser Ser Gly
115 120 125
Ser Ser Met Pro Ser Leu Leu Gln Cys Leu Lys Ala Pro Val Leu Ser
130 135 140
Asp Ser Ser Cys Lys Ser Ser Tyr Pro Gly Gln Ile Thr Gly Asn Met
145 150 155 160
Ile Cys Val Gly Phe Leu Glu Gly Gly Ala Asp Ala Cys Gln Gly Asp
165 170 175
Ser Gly Gly Pro Val Val Cys Asn Gly Gln Leu Gln Gly Ile Val Ser
180 185 190
Ser Gly Tyr Gly Cys Ala Gln Lys Asn Lys Pro Gly Val Tyr Thr Lys
195 200 205
Val Cys Asn Tyr Val Asn Trp Ile Gln Gln Thr Ile Ala Ala Asn
210 215 220
<210> 105
<211> 223
<212> PRT
<213> 人工序列
<220>
<223> 猪胰蛋白酶变体105号
<400> 105
Ile Val Gly Gly Tyr Thr Cys Ala Ala Asn Ser Ile Pro Tyr Gln Val
1 5 10 15
Ser Leu Asn Ser Gly Ser His Arg Cys Gly Gly Ser Leu Ile Asn Ser
20 25 30
Gln Trp Val Val Ser Ala Ala His Cys Tyr Lys Ser Arg Ile Gln Val
35 40 45
Arg Leu Gly Glu His Asn Ile His Val Leu Glu Gly Asn Glu Gln Phe
50 55 60
Ile Asn Ala Ala Lys Ile Ile Thr His Pro Asn Phe Asn Pro Asn Thr
65 70 75 80
Leu Asp Asn Asp Ile Met Leu Ile Lys Leu Ser Ser Pro Ala Thr Leu
85 90 95
Asn Ser Asn Val Ala Thr Val Ser Leu Pro Thr Ser Cys Ala Ala Ala
100 105 110
Gly Thr Glu Cys Leu Ile Ser Gly Trp Gly Asn Thr Ala Ser Ser Gly
115 120 125
Ser Ser Tyr Pro Ser Leu Leu Gln Cys Leu Lys Ala Pro Val Leu Ser
130 135 140
Asp Ser Ser Cys Lys Ser Ser Tyr Pro Gly Gln Ile Thr Gly Asn Met
145 150 155 160
Ile Cys Val Gly Phe Leu Glu Gly Gly Ala Asp Ala Cys Gln Gly Asp
165 170 175
Ser Gly Gly Pro Val Val Cys Asn Gly Gln Leu Gln Gly Ile Val Ser
180 185 190
Ser Gly Tyr Gly Cys Ala Gln Lys Asn Lys Pro Gly Val Tyr Thr Lys
195 200 205
Val Cys Asn Tyr Val Asn Trp Ile Gln Gln Thr Ile Ala Ala Asn
210 215 220
<210> 106
<211> 223
<212> PRT
<213> 人工序列
<220>
<223> 猪胰蛋白酶变体106号
<400> 106
Ile Val Gly Gly Tyr Thr Cys Ala Ala Asn Ser Ile Pro Tyr Gln Val
1 5 10 15
Ser Leu Asn Ser Gly Ser His Arg Cys Gly Gly Ser Leu Ile Asn Ser
20 25 30
Gln Trp Val Val Ser Ala Ala His Cys Tyr Lys Ser Arg Ile Gln Val
35 40 45
Arg Leu Gly Glu His Asn Ile His Val Leu Glu Gly Asn Glu Gln Phe
50 55 60
Ile Asn Ala Ala Lys Ile Ile Thr His Pro Asn Phe Asn Pro Asn Thr
65 70 75 80
Leu Asp Asn Asp Ile Met Leu Ile Lys Leu Ser Ser Pro Ala Thr Leu
85 90 95
Asn Ser Ala Val Ala Thr Val Ser Leu Pro Thr Ser Cys Ala Ala Ala
100 105 110
Gly Thr Glu Cys Leu Ile Ser Gly Trp Gly Asn Thr Cys Ser Ser Gly
115 120 125
Ser Ser Met Pro Ser Leu Leu Gln Cys Leu Lys Ala Pro Val Leu Ser
130 135 140
Asp Ser Ser Cys Lys Ser Ser Tyr Pro Gly Gln Ile Thr Gly Asn Met
145 150 155 160
Ile Cys Val Gly Phe Leu Glu Gly Gly Ala Asp Ala Cys Gln Gly Asp
165 170 175
Ser Gly Gly Pro Val Val Cys Asn Gly Gln Leu Gln Gly Ile Val Ser
180 185 190
Ser Gly Tyr Gly Cys Ala Gln Lys Asn Lys Pro Gly Val Tyr Thr Lys
195 200 205
Val Cys Asn Tyr Val Asn Trp Ile Gln Gln Thr Ile Ala Ala Asn
210 215 220
<210> 107
<211> 223
<212> PRT
<213> 人工序列
<220>
<223> 猪胰蛋白酶变体107号
<400> 107
Ile Val Gly Gly Tyr Thr Cys Ala Ala Asn Ser Ile Pro Tyr Gln Val
1 5 10 15
Ser Leu Asn Ser Gly Ser His Arg Cys Gly Gly Ser Leu Ile Asn Ser
20 25 30
Gln Trp Val Val Ser Ala Ala His Cys Tyr Lys Pro Arg Ile Gln Val
35 40 45
Arg Leu Gly Glu His Asn Ile His Val Leu Glu Gly Asn Glu Gln Phe
50 55 60
Ile Asn Ala Ala Lys Ile Ile Thr His Pro Asn Phe Asn Pro Asn Thr
65 70 75 80
Leu Asp Asn Asp Ile Met Leu Ile Lys Leu Ser Ser Pro Ala Thr Leu
85 90 95
Asn Ser His Val Ala Thr Val Ser Leu Pro Thr Ser Cys Ala Ala Ala
100 105 110
Gly Thr Glu Cys Leu Ile Ser Gly Trp Gly Asn Thr Cys Ser Ser Gly
115 120 125
Ser Ser Met Pro Ser Leu Leu Gln Cys Leu Lys Ala Pro Val Leu Ser
130 135 140
Asp Ser Ser Cys Lys Ser Ser Tyr Pro Gly Gln Ile Thr Gly Asn Met
145 150 155 160
Ile Cys Val Gly Phe Leu Glu Gly Gly Ala Asp Ala Cys Gln Gly Asp
165 170 175
Ser Gly Gly Pro Val Val Cys Asn Gly Gln Leu Gln Gly Ile Val Ser
180 185 190
Ser Gly Tyr Gly Cys Ala Gln Lys Asn Lys Pro Gly Val Tyr Thr Lys
195 200 205
Val Cys Asn Tyr Val Asn Trp Ile Gln Gln Thr Ile Ala Ala Asn
210 215 220
<210> 108
<211> 223
<212> PRT
<213> 人工序列
<220>
<223> 猪胰蛋白酶变体108号
<400> 108
Ile Val Gly Gly Tyr Thr Cys Ala Ala Asn Ser Ile Pro Tyr Gln Val
1 5 10 15
Ser Leu Asn Ser Gly Ser His Arg Cys Gly Gly Ser Leu Ile Asn Ser
20 25 30
Gln Trp Val Val Ser Ala Ala His Cys Tyr Lys Pro Arg Ile Gln Val
35 40 45
Arg Leu Gly Glu His Asn Ile Asp Val Leu Glu Gly Asn Glu Gln Phe
50 55 60
Ile Asn Ala Ala Lys Ile Ile Thr His Pro Asn Phe Asn Pro Asn Thr
65 70 75 80
Leu Asp Asn Asp Ile Met Leu Ile Lys Leu Ser Ser Pro Ala Thr Leu
85 90 95
Asn Ser Ala Val Ala Thr Val Ser Leu Pro Thr Ser Cys Ala Ala Ala
100 105 110
Gly Thr Glu Cys Leu Ile Ser Gly Trp Gly Asn Thr Cys Ser Ser Gly
115 120 125
Ser Ser Met Pro Ser Leu Leu Gln Cys Leu Lys Ala Pro Val Leu Ser
130 135 140
Asp Ser Ser Cys Lys Ser Ser Tyr Pro Gly Gln Ile Thr Gly Asn Met
145 150 155 160
Ile Cys Val Gly Phe Leu Glu Gly Gly Ala Asp Ala Cys Gln Gly Asp
165 170 175
Ser Gly Gly Pro Val Val Cys Asn Gly Gln Leu Gln Gly Ile Val Ser
180 185 190
Ser Gly Tyr Gly Cys Ala Gln Lys Asn Lys Pro Gly Val Tyr Thr Lys
195 200 205
Val Cys Asn Tyr Val Asn Trp Ile Gln Gln Thr Ile Ala Ala Asn
210 215 220
<210> 109
<211> 223
<212> PRT
<213> 人工序列
<220>
<223> 猪胰蛋白酶变体109号
<400> 109
Ile Val Gly Gly Tyr Thr Cys Ala Ala Asn Ser Ile Pro Tyr Gln Val
1 5 10 15
Ser Leu Asn Ser Gly Ser His Arg Cys Gly Gly Ser Leu Ile Asn Ser
20 25 30
Gln Trp Val Val Ser Ala Ala His Cys Tyr Lys Ser Arg Ile Gln Val
35 40 45
Arg Leu Gly Glu His Asn Ile His Val Leu Glu Gly Asn Glu Gln Phe
50 55 60
Ile Asn Ala Ala Lys Ile Ile Thr His Pro Asn Phe Asn Gly Asn Thr
65 70 75 80
Leu Asp Asn Asp Ile Met Leu Ile Lys Leu Ser Ser Pro Ala Thr Leu
85 90 95
Asn Ser Asn Val Ala Thr Val Ser Leu Pro Thr Ser Cys Ala Ala Ala
100 105 110
Gly Thr Glu Cys Leu Ile Ser Gly Trp Gly Asn Thr Cys Ser Ser Gly
115 120 125
Ser Ser Met Pro Ser Leu Leu Gln Cys Leu Lys Ala Pro Val Leu Ser
130 135 140
Asp Ser Ser Cys Lys Ser Ser Tyr Pro Gly Gln Ile Thr Gly Asn Met
145 150 155 160
Ile Cys Val Gly Phe Leu Glu Gly Gly Ala Asp Ala Cys Gln Gly Asp
165 170 175
Ser Gly Gly Pro Val Val Cys Asn Gly Gln Leu Gln Gly Ile Val Ser
180 185 190
Ser Gly Tyr Gly Cys Ala Gln Lys Asn Lys Pro Gly Val Tyr Thr Lys
195 200 205
Val Cys Asn Tyr Val Asn Trp Ile Gln Gln Thr Ile Ala Ala Asn
210 215 220
<210> 110
<211> 223
<212> PRT
<213> 人工序列
<220>
<223> 猪胰蛋白酶变体110号
<400> 110
Ile Val Gly Gly Tyr Thr Cys Ala Ala Asn Ser Ile Pro Tyr Gln Val
1 5 10 15
Ser Leu Asn Ser Gly Ser His Arg Cys Gly Gly Ser Leu Ile Asn Ser
20 25 30
Gln Trp Val Val Ser Ala Ala His Cys Tyr Lys Ser Arg Ile Gln Val
35 40 45
Arg Leu Gly Glu His Asn Ile His Val Leu Glu Gly Asn Glu Gln Phe
50 55 60
Ile Asn Ala Ala Lys Ile Ile Thr His Pro Asn Phe Asn Pro Asn Thr
65 70 75 80
Leu Asp Asn Asp Ile Met Leu Ile Lys Leu Ser Ser Pro Ala Thr Leu
85 90 95
Asn Ser His Val Ala Thr Val Ser Leu Pro Arg Ser Cys Ala Ala Ala
100 105 110
Gly Thr Glu Cys Leu Ile Ser Gly Trp Gly Asn Thr Cys Ser Ser Gly
115 120 125
Ser Ser Met Pro Ser Leu Leu Gln Cys Leu Lys Ala Pro Val Leu Ser
130 135 140
Asp Ser Ser Cys Lys Ser Ser Tyr Pro Gly Gln Ile Thr Gly Asn Met
145 150 155 160
Ile Cys Val Gly Phe Leu Glu Gly Gly Ala Asp Ala Cys Gln Gly Asp
165 170 175
Ser Gly Gly Pro Val Val Cys Asn Gly Gln Leu Gln Gly Ile Val Ser
180 185 190
Ser Gly Tyr Gly Cys Ala Gln Lys Asn Lys Pro Gly Val Tyr Thr Lys
195 200 205
Val Cys Asn Tyr Val Asn Trp Ile Gln Gln Thr Ile Ala Ala Asn
210 215 220
<210> 111
<211> 223
<212> PRT
<213> 人工序列
<220>
<223> 猪胰蛋白酶变体111号
<400> 111
Ile Val Gly Gly Tyr Thr Cys Ala Ala Asn Ser Ile Pro Tyr Gln Val
1 5 10 15
Ser Leu Asn Ser Gly Ser His Arg Cys Gly Gly Ser Leu Ile Asn Ser
20 25 30
Gln Trp Val Val Ser Ala Ala His Cys Tyr Lys Pro Arg Ile Gln Val
35 40 45
Arg Leu Gly Glu His Asn Ile His Val Leu Glu Gly Asn Glu Gln Phe
50 55 60
Ile Asn Ala Ala Lys Ile Ile Thr His Pro Asn Phe Asn Pro Asn Thr
65 70 75 80
Leu Asp Asn Asp Ile Met Leu Ile Lys Leu Ser Ser Pro Ala Thr Leu
85 90 95
Asn Ser His Val Ala Thr Val Ser Leu Pro Thr Ser Cys Ala Ala Ala
100 105 110
Gly Thr Glu Cys Leu Ile Ser Gly Trp Gly Asn Thr Ser Ser Ser Gly
115 120 125
Ser Ser Tyr Pro Ser Leu Leu Gln Cys Leu Lys Ala Pro Val Leu Ser
130 135 140
Asp Ser Ser Cys Lys Ser Ser Tyr Pro Gly Gln Ile Thr Gly Asn Met
145 150 155 160
Ile Cys Val Gly Phe Leu Glu Gly Gly Ala Asp Ala Cys Gln Gly Asp
165 170 175
Ser Gly Gly Pro Val Val Cys Asn Gly Gln Leu Gln Gly Ile Val Ser
180 185 190
Ser Gly Tyr Gly Cys Ala Gln Lys Asn Lys Pro Gly Val Tyr Thr Lys
195 200 205
Val Cys Asn Tyr Val Asn Trp Ile Gln Gln Thr Ile Ala Ala Asn
210 215 220
<210> 112
<211> 223
<212> PRT
<213> 人工序列
<220>
<223> 猪胰蛋白酶变体112号
<400> 112
Ile Val Gly Gly Tyr Thr Cys Ala Ala Asn Ser Ile Pro Tyr Gln Val
1 5 10 15
Ser Leu Asn Ser Gly Ser His Arg Cys Gly Gly Ser Leu Ile Asn Ser
20 25 30
Gln Trp Val Val Ser Ala Ala His Cys Tyr Lys Pro Arg Ile Gln Val
35 40 45
Arg Leu Gly Glu His Asn Ile His Val Leu Glu Gly Asn Glu Gln Phe
50 55 60
Ile Asn Ala Ala Lys Ile Ile Thr His Pro Asn Phe Asn Pro Asn Thr
65 70 75 80
Leu Asp Asn Asp Ile Met Leu Ile Lys Leu Ser Ser Pro Ala Thr Leu
85 90 95
Asn Ser His Val Ala Thr Val Ser Leu Pro Thr Ser Cys Ala Ala Ala
100 105 110
Gly Thr Glu Cys Leu Ile Ser Gly Trp Gly Asn Thr Ser Ser Ser Gly
115 120 125
Ser Ser Tyr Pro Ser Leu Leu Gln Cys Leu Lys Ala Pro Val Leu Ser
130 135 140
Asp Ser Ser Cys Lys Ser Ser Tyr Pro Gly Gln Ile Thr Gly Asn Met
145 150 155 160
Ile Cys Val Gly Phe Leu Glu Gly Gly Ala Asp Ser Cys Gln Gly Asp
165 170 175
Ser Gly Gly Pro Val Val Cys Asn Gly Gln Leu Gln Gly Ile Val Ser
180 185 190
Ser Gly Tyr Gly Cys Ala Gln Lys Asn Lys Pro Gly Val Tyr Thr Lys
195 200 205
Val Cys Asn Tyr Val Asn Trp Ile Gln Gln Thr Ile Ala Ala Asn
210 215 220
<210> 113
<211> 223
<212> PRT
<213> 人工序列
<220>
<223> 猪胰蛋白酶变体113号
<400> 113
Ile Val Gly Gly Tyr Thr Cys Ala Ala Asn Ser Ile Pro Tyr Gln Val
1 5 10 15
Ser Leu Asn Ser Gly Ser His Arg Cys Gly Gly Ser Leu Ile Asn Ser
20 25 30
Gln Trp Val Val Ser Ala Ala His Cys Tyr Lys Pro Arg Ile Gln Val
35 40 45
Arg Leu Gly Glu His Asn Ile His Val Leu Glu Gly Asn Glu Gln Phe
50 55 60
Ile Asn Ala Ala Lys Ile Ile Thr His Pro Asn Phe Asn Gly Asn Thr
65 70 75 80
Leu Asp Asn Asp Ile Met Leu Ile Lys Leu Ser Ser Pro Ala Thr Leu
85 90 95
Asn Ser Ala Val Ala Thr Val Ser Leu Pro Thr Ser Cys Ala Ala Ala
100 105 110
Gly Thr Glu Cys Leu Ile Ser Gly Trp Gly Asn Thr Ala Ser Ser Gly
115 120 125
Ser Ser Tyr Pro Ser Leu Leu Gln Cys Leu Lys Ala Pro Val Leu Ser
130 135 140
Asp Ser Ser Cys Lys Ser Ser Tyr Pro Gly Gln Ile Thr Gly Asn Met
145 150 155 160
Ile Cys Val Gly Phe Leu Glu Gly Gly Ala Asp Ala Cys Gln Gly Asp
165 170 175
Ser Gly Gly Pro Val Val Cys Asn Gly Gln Leu Gln Gly Ile Val Ser
180 185 190
Ser Gly Tyr Gly Cys Ala Gln Lys Asn Lys Pro Gly Val Tyr Thr Lys
195 200 205
Val Cys Asn Tyr Val Asn Trp Ile Gln Gln Thr Ile Ala Ala Asn
210 215 220
<210> 114
<211> 223
<212> PRT
<213> 人工序列
<220>
<223> 猪胰蛋白酶变体114号
<400> 114
Ile Val Gly Gly Tyr Thr Cys Ala Ala Asn Ser Ile Pro Tyr Gln Val
1 5 10 15
Ser Leu Asn Ser Gly Ser His Arg Cys Gly Gly Ser Leu Ile Asn Ser
20 25 30
Gln Trp Val Val Ser Ala Ala His Cys Tyr Lys Pro Arg Ile Gln Val
35 40 45
Arg Leu Gly Glu His Asn Ile His Val Leu Glu Gly Asn Glu Gln Phe
50 55 60
Ile Asn Ala Ala Lys Ile Ile Thr His Pro Asn Phe Asn Gly Asn Thr
65 70 75 80
Leu Asp Asn Asp Ile Met Leu Ile Lys Leu Ser Ser Pro Ala Thr Leu
85 90 95
Asn Ser Ala Val Ala Thr Val Ser Leu Pro Thr Ser Cys Ala Ala Ala
100 105 110
Gly Thr Glu Cys Leu Ile Ser Gly Trp Gly Asn Thr Ala Ser Ser Gly
115 120 125
Ser Ser Tyr Pro Ser Leu Leu Gln Cys Leu Lys Ala Pro Val Leu Ser
130 135 140
Asp Ser Ser Cys Lys Ser Ser Tyr Pro Gly Gln Ile Thr Gly Asn Met
145 150 155 160
Ile Cys Val Gly Phe Leu Glu Gly Gly Ala Asp Ser Cys Gln Gly Asp
165 170 175
Ser Gly Gly Pro Val Val Cys Asn Gly Gln Leu Gln Gly Ile Val Ser
180 185 190
Ser Gly Tyr Gly Cys Ala Gln Lys Asn Lys Pro Gly Val Tyr Thr Lys
195 200 205
Val Cys Asn Tyr Val Asn Trp Ile Gln Gln Thr Ile Ala Ala Asn
210 215 220
<210> 115
<211> 223
<212> PRT
<213> 人工序列
<220>
<223> 猪胰蛋白酶变体115号
<400> 115
Ile Val Gly Gly Tyr Thr Cys Ala Ala Asn Ser Ile Pro Tyr Gln Val
1 5 10 15
Ser Leu Asn Ser Gly Ser His Arg Cys Gly Gly Ser Leu Ile Asn Ser
20 25 30
Gln Trp Val Val Ser Ala Ala His Cys Tyr Lys Pro Arg Ile Gln Val
35 40 45
Arg Leu Gly Glu His Asn Ile His Val Leu Glu Gly Asn Glu Gln Phe
50 55 60
Ile Asn Ala Ala Lys Ile Ile Thr His Pro Asn Phe Asn Gly Asn Thr
65 70 75 80
Leu Asp Asn Asp Ile Met Leu Ile Lys Leu Ser Ser Pro Ala Thr Leu
85 90 95
Asn Ser Asn Val Ala Thr Val Ser Leu Pro Thr Ser Cys Ala Ala Ala
100 105 110
Gly Thr Glu Cys Leu Ile Ser Gly Trp Gly Asn Thr Ser Ser Ser Gly
115 120 125
Ser Ser Met Pro Ser Leu Leu Gln Cys Leu Lys Ala Pro Val Leu Ser
130 135 140
Asp Ser Ser Cys Lys Ser Ser Tyr Pro Gly Gln Ile Thr Gly Asn Met
145 150 155 160
Ile Cys Val Gly Phe Leu Glu Gly Gly Lys Asp Ala Cys Gln Gly Asp
165 170 175
Ser Gly Gly Pro Val Val Cys Asn Gly Gln Leu Gln Gly Ile Val Ser
180 185 190
Ser Gly Tyr Gly Cys Ala Gln Lys Asn Lys Pro Gly Val Tyr Thr Lys
195 200 205
Val Cys Asn Tyr Val Asn Trp Ile Gln Gln Thr Ile Ala Ala Asn
210 215 220
<210> 116
<211> 223
<212> PRT
<213> 人工序列
<220>
<223> 猪胰蛋白酶变体116号
<400> 116
Ile Val Gly Gly Tyr Thr Cys Ala Ala Asn Ser Ile Pro Tyr Gln Val
1 5 10 15
Ser Leu Asn Ser Gly Ser His Arg Cys Gly Gly Ser Leu Ile Asn Ser
20 25 30
Gln Trp Val Val Ser Ala Ala His Cys Tyr Lys Pro Arg Ile Gln Val
35 40 45
Arg Leu Gly Glu His Asn Ile His Val Leu Glu Gly Asn Glu Gln Phe
50 55 60
Ile Asn Ala Ala Lys Ile Ile Thr His Pro Asn Phe Asn Gly Asn Thr
65 70 75 80
Leu Asp Asn Asp Ile Met Leu Ile Lys Leu Ser Ser Pro Ala Thr Leu
85 90 95
Asn Ser Asn Val Ala Thr Val Ser Leu Pro Thr Ser Cys Ala Ala Ala
100 105 110
Gly Thr Glu Cys Leu Ile Ser Gly Trp Gly Asn Thr Ser Ser Ser Gly
115 120 125
Ser Ser Met Pro Ser Leu Leu Gln Cys Leu Lys Ala Pro Val Leu Ser
130 135 140
Asp Ser Ser Cys Lys Ser Ser Tyr Pro Gly Gln Ile Thr Gly Asn Met
145 150 155 160
Ile Cys Val Gly Phe Leu Glu Gly Gly Ala Asp Ala Cys Gln Gly Asp
165 170 175
Ser Gly Gly Pro Val Val Cys Asn Gly Gln Leu Gln Gly Ile Val Ser
180 185 190
Ser Gly Tyr Gly Cys Ala Gln Lys Asn Lys Pro Gly Val Tyr Thr Lys
195 200 205
Val Cys Asn Tyr Val Asn Trp Ile Gln Gln Thr Ile Ala Ala Asn
210 215 220
<210> 117
<211> 223
<212> PRT
<213> 人工序列
<220>
<223> 猪胰蛋白酶变体117号
<400> 117
Ile Val Gly Gly Tyr Thr Cys Ala Ala Asn Ser Ile Pro Tyr Gln Val
1 5 10 15
Ser Leu Asn Ser Gly Ser His Arg Cys Gly Gly Ser Leu Ile Asn Ser
20 25 30
Gln Trp Val Val Ser Ala Ala His Cys Tyr Lys Pro Arg Ile Gln Val
35 40 45
Arg Leu Gly Glu His Asn Ile His Val Leu Glu Gly Asn Glu Gln Phe
50 55 60
Ile Asn Ala Ala Lys Ile Ile Thr His Pro Asn Phe Asn Gly Asn Thr
65 70 75 80
Leu Asp Asn Asp Ile Met Leu Ile Lys Leu Ser Ser Pro Ala Thr Leu
85 90 95
Asn Ser Asn Val Ala Thr Val Ser Leu Pro Thr Ser Cys Ala Ala Ala
100 105 110
Gly Thr Glu Cys Leu Ile Ser Gly Trp Gly Asn Thr Ala Ser Ser Gly
115 120 125
Ser Ser Met Pro Ser Leu Leu Gln Cys Leu Lys Ala Pro Val Leu Ser
130 135 140
Asp Ser Ser Cys Lys Ser Ser Tyr Pro Gly Gln Ile Thr Gly Asn Met
145 150 155 160
Ile Cys Val Gly Phe Leu Glu Gly Gly Lys Asp Ala Cys Gln Gly Asp
165 170 175
Ser Gly Gly Pro Val Val Cys Asn Gly Gln Leu Gln Gly Ile Val Ser
180 185 190
Ser Gly Tyr Gly Cys Ala Gln Lys Asn Lys Pro Gly Val Tyr Thr Lys
195 200 205
Val Cys Asn Tyr Val Asn Trp Ile Gln Gln Thr Ile Ala Ala Asn
210 215 220
<210> 118
<211> 223
<212> PRT
<213> 人工序列
<220>
<223> 猪胰蛋白酶变体118号
<400> 118
Ile Val Gly Gly Tyr Thr Cys Ala Ala Asn Ser Ile Pro Tyr Gln Val
1 5 10 15
Ser Leu Asn Ser Gly Ser His Arg Cys Gly Gly Ser Leu Ile Asn Ser
20 25 30
Gln Trp Val Val Ser Ala Ala His Cys Tyr Lys Pro Arg Ile Gln Val
35 40 45
Arg Leu Gly Glu His Asn Ile His Val Leu Glu Gly Asn Glu Gln Phe
50 55 60
Ile Asn Ala Ala Lys Ile Ile Thr His Pro Asn Phe Asn Gly Asn Thr
65 70 75 80
Leu Asp Asn Asp Ile Met Leu Ile Lys Leu Ser Ser Pro Ala Thr Leu
85 90 95
Asn Ser His Val Ala Thr Val Ser Leu Pro Thr Ser Cys Ala Ala Ala
100 105 110
Gly Thr Glu Cys Leu Ile Ser Gly Trp Gly Asn Thr Ala Ser Ser Gly
115 120 125
Ser Ser Met Pro Ser Leu Leu Gln Cys Leu Lys Ala Pro Val Leu Ser
130 135 140
Asp Ser Ser Cys Lys Ser Ser Tyr Pro Gly Gln Ile Thr Gly Asn Met
145 150 155 160
Ile Cys Val Gly Phe Leu Glu Gly Gly Ala Asp Ala Cys Gln Gly Asp
165 170 175
Ser Gly Gly Pro Val Val Cys Asn Gly Gln Leu Gln Gly Ile Val Ser
180 185 190
Ser Gly Tyr Gly Cys Ala Gln Lys Asn Lys Pro Gly Val Tyr Thr Lys
195 200 205
Val Cys Asn Tyr Val Asn Trp Ile Gln Gln Thr Ile Ala Ala Asn
210 215 220
<210> 119
<211> 223
<212> PRT
<213> 人工序列
<220>
<223> 猪胰蛋白酶变体119号
<400> 119
Ile Val Gly Gly Tyr Thr Cys Ala Ala Asn Ser Ile Pro Tyr Gln Val
1 5 10 15
Ser Leu Asn Ser Gly Ser His Arg Cys Gly Gly Ser Leu Ile Asn Ser
20 25 30
Gln Trp Val Val Ser Ala Ala His Cys Tyr Lys Pro Arg Ile Gln Val
35 40 45
Arg Leu Gly Glu His Asn Ile His Val Leu Glu Gly Asn Glu Gln Phe
50 55 60
Ile Asn Ala Ala Lys Ile Ile Thr His Pro Asn Phe Asn Gly Asn Thr
65 70 75 80
Leu Asp Asn Asp Ile Met Leu Ile Lys Leu Ser Ser Pro Ala Thr Leu
85 90 95
Asn Ser Ala Val Ala Thr Val Ser Leu Pro Thr Ser Cys Ala Ala Ala
100 105 110
Gly Thr Glu Cys Leu Ile Ser Gly Trp Gly Asn Thr Ala Ser Ser Gly
115 120 125
Ser Ser Met Pro Ser Leu Leu Gln Cys Leu Lys Ala Pro Val Leu Ser
130 135 140
Asp Ser Ser Cys Lys Ser Ser Tyr Pro Gly Gln Ile Thr Gly Asn Met
145 150 155 160
Ile Cys Val Gly Phe Leu Glu Gly Gly Lys Asp Ala Cys Gln Gly Asp
165 170 175
Ser Gly Gly Pro Val Val Cys Asn Gly Gln Leu Gln Gly Ile Val Ser
180 185 190
Ser Gly Tyr Gly Cys Ala Gln Lys Asn Lys Pro Gly Val Tyr Thr Lys
195 200 205
Val Cys Asn Tyr Val Asn Trp Ile Gln Gln Thr Ile Ala Ala Asn
210 215 220
<210> 120
<211> 223
<212> PRT
<213> 人工序列
<220>
<223> 猪胰蛋白酶变体120号
<400> 120
Ile Val Gly Gly Tyr Thr Cys Ala Ala Asn Ser Ile Pro Tyr Gln Val
1 5 10 15
Ser Leu Asn Ser Gly Ser His Arg Cys Gly Gly Ser Leu Ile Asn Ser
20 25 30
Gln Trp Val Val Ser Ala Ala His Cys Tyr Lys Ser Arg Ile Gln Val
35 40 45
Arg Leu Gly Glu His Asn Ile Asp Val Leu Glu Gly Asn Glu Gln Phe
50 55 60
Ile Asn Ala Ala Lys Ile Ile Thr His Pro Asn Phe Asn Gly Asn Thr
65 70 75 80
Leu Asp Asn Asp Ile Met Leu Ile Lys Leu Ser Ser Pro Ala Thr Leu
85 90 95
Asn Ser Arg Val Ala Thr Val Ser Leu Pro Arg Ser Cys Ala Ala Ala
100 105 110
Gly Thr Glu Cys Leu Ile Ser Gly Trp Gly Asn Thr Pro Ser Ser Gly
115 120 125
Ser Ser Tyr Pro Ser Leu Leu Gln Cys Leu Lys Ala Pro Val Leu Ser
130 135 140
Asp Ser Ser Cys Lys Ser Ser Tyr Pro Gly Gln Ile Thr Gly Asn Met
145 150 155 160
Ile Cys Val Gly Phe Leu Glu Gly Gly Lys Asp Ala Cys Gln Gly Asp
165 170 175
Ser Gly Gly Pro Val Val Cys Asn Gly Gln Leu Gln Gly Ile Val Ser
180 185 190
Ser Gly Tyr Gly Cys Ala Gln Lys Asn Lys Pro Gly Val Tyr Thr Lys
195 200 205
Val Cys Asn Tyr Val Asn Trp Ile Gln Gln Thr Ile Ala Ala Asn
210 215 220
<210> 121
<211> 223
<212> PRT
<213> 人工序列
<220>
<223> 猪胰蛋白酶变体121号
<400> 121
Ile Val Gly Gly Tyr Thr Cys Ala Ala Asn Ser Ile Pro Tyr Gln Val
1 5 10 15
Ser Leu Asn Ser Gly Ser His Arg Cys Gly Gly Ser Leu Ile Asn Ser
20 25 30
Gln Trp Val Val Ser Ala Ala His Cys Tyr Lys Ser Arg Ile Gln Val
35 40 45
Arg Leu Gly Glu His Asn Ile Asp Val Leu Glu Gly Asn Glu Gln Phe
50 55 60
Ile Asn Ala Ala Lys Ile Ile Thr His Pro Asn Phe Asn Gly Asn Thr
65 70 75 80
Leu Asp Asn Asp Ile Met Leu Ile Lys Leu Ser Ser Pro Ala Thr Leu
85 90 95
Asn Ser Arg Val Ala Thr Val Ser Leu Pro Arg Ser Cys Ala Ala Ala
100 105 110
Gly Thr Glu Cys Leu Ile Ser Gly Trp Gly Asn Thr Lys Ser Ser Gly
115 120 125
Ser Ser Tyr Pro Ser Leu Leu Gln Cys Leu Lys Ala Pro Val Leu Ser
130 135 140
Asp Ser Ser Cys Lys Ser Ser Tyr Pro Gly Gln Ile Thr Gly Asn Met
145 150 155 160
Ile Cys Val Gly Phe Leu Glu Gly Gly Ile Asp Ala Cys Gln Gly Asp
165 170 175
Ser Gly Gly Pro Val Val Cys Asn Gly Gln Leu Gln Gly Ile Val Ser
180 185 190
Ser Gly Tyr Gly Cys Ala Gln Lys Asn Lys Pro Gly Val Tyr Thr Lys
195 200 205
Val Cys Asn Tyr Val Asn Trp Ile Gln Gln Thr Ile Ala Ala Asn
210 215 220
<210> 122
<211> 223
<212> PRT
<213> 人工序列
<220>
<223> 猪胰蛋白酶变体122号
<400> 122
Ile Val Gly Gly Tyr Thr Cys Ala Ala Asn Ser Ile Pro Tyr Gln Val
1 5 10 15
Ser Leu Asn Ser Gly Ser His Arg Cys Gly Gly Ser Leu Ile Asn Ser
20 25 30
Gln Trp Val Val Ser Ala Ala His Cys Tyr Lys Ser Arg Ile Gln Val
35 40 45
Arg Leu Gly Glu His Asn Ile Asp Val Leu Glu Gly Asn Glu Gln Phe
50 55 60
Ile Asn Ala Ala Lys Ile Ile Thr His Pro Asn Phe Asn Gly Asn Thr
65 70 75 80
Leu Asp Asn Asp Ile Met Leu Ile Lys Leu Ser Ser Pro Ala Thr Leu
85 90 95
Asn Ser Arg Val Ala Thr Val Ser Leu Pro Arg Ser Cys Ala Ala Ala
100 105 110
Gly Thr Glu Cys Leu Ile Ser Gly Trp Gly Asn Thr Lys Ser Ser Gly
115 120 125
Ser Ser Tyr Pro Ser Leu Leu Gln Cys Leu Lys Ala Pro Val Leu Ser
130 135 140
Asp Ser Ser Cys Lys Ser Ser Tyr Pro Gly Gln Ile Thr Gly Asn Met
145 150 155 160
Ile Cys Val Gly Phe Leu Glu Gly Gly Phe Asp Ala Cys Gln Gly Asp
165 170 175
Ser Gly Gly Pro Val Val Cys Asn Gly Gln Leu Gln Gly Ile Val Ser
180 185 190
Ser Gly Tyr Gly Cys Ala Gln Lys Asn Lys Pro Gly Val Tyr Thr Lys
195 200 205
Val Cys Asn Tyr Val Asn Trp Ile Gln Gln Thr Ile Ala Ala Asn
210 215 220
<210> 123
<211> 223
<212> PRT
<213> 人工序列
<220>
<223> 猪胰蛋白酶变体123号
<400> 123
Ile Val Gly Gly Tyr Thr Cys Ala Ala Asn Ser Ile Pro Tyr Gln Val
1 5 10 15
Ser Leu Asn Ser Gly Ser His Gly Cys Gly Gly Ser Leu Ile Asn Ser
20 25 30
Gln Trp Val Val Ser Ala Ala His Cys Tyr Lys Ser Arg Ile Gln Val
35 40 45
Arg Leu Gly Glu His Asn Ile Asp Val Leu Glu Gly Asn Glu Gln Phe
50 55 60
Ile Asn Ala Ala Lys Ile Ile Thr His Pro Asn Phe Asn Gly Asn Thr
65 70 75 80
Leu Asp Asn Asp Ile Met Leu Ile Lys Leu Ser Ser Pro Ala Thr Leu
85 90 95
Asn Ser Arg Val Ala Thr Val Ser Leu Pro Arg Ser Cys Ala Ala Ala
100 105 110
Gly Thr Glu Cys Leu Ile Ser Gly Trp Gly Asn Thr Lys Ser Ser Gly
115 120 125
Ser Ser Tyr Pro Ser Leu Leu Gln Cys Leu Lys Ala Pro Val Leu Ser
130 135 140
Asp Ser Ser Cys Lys Ser Ser Tyr Pro Gly Gln Ile Thr Gly Asn Met
145 150 155 160
Ile Cys Val Gly Phe Leu Glu Gly Gly Lys Asp Ala Cys Gln Gly Asp
165 170 175
Ser Gly Gly Pro Val Val Cys Asn Gly Gln Leu Gln Gly Ile Val Ser
180 185 190
Ser Gly Tyr Gly Cys Ala Gln Lys Asn Lys Pro Gly Val Tyr Thr Lys
195 200 205
Val Cys Asn Tyr Val Asn Trp Ile Gln Gln Thr Ile Ala Ala Asn
210 215 220
<210> 124
<211> 223
<212> PRT
<213> 人工序列
<220>
<223> 猪胰蛋白酶变体124号
<400> 124
Ile Val Gly Gly Tyr Thr Cys Ala Ala Asn Ser Ile Pro Tyr Gln Val
1 5 10 15
Ser Leu Asn Ser Gly Ser His Arg Cys Gly Gly Ser Leu Ile Asn Ser
20 25 30
Gln Trp Val Val Ser Ala Ala His Cys Tyr Lys Ser Arg Ile Gln Val
35 40 45
Arg Leu Gly Glu His Asn Ile Asp Val Leu Glu Gly Asn Glu Gln Phe
50 55 60
Ile Asn Ala Ala Lys Ile Ile Thr His Pro Asn Phe Asn Gly Asn Thr
65 70 75 80
Leu Asp Asn Asp Ile Met Leu Ile Lys Leu Ser Ser Pro Ala Thr Leu
85 90 95
Asn Ser Arg Val Ala Thr Val Ser Leu Pro Arg Ser Cys Ala Ala Ala
100 105 110
Gly Thr Glu Cys Leu Ile Ser Gly Trp Gly Asn Thr Lys Ser Ser Gly
115 120 125
Ser Ser Lys Pro Ser Leu Leu Gln Cys Leu Lys Ala Pro Val Leu Ser
130 135 140
Asp Ser Ser Cys Lys Ser Ser Tyr Pro Gly Gln Ile Thr Gly Asn Met
145 150 155 160
Ile Cys Val Gly Phe Leu Glu Gly Gly Lys Asp Ala Cys Gln Gly Asp
165 170 175
Ser Gly Gly Pro Val Val Cys Asn Gly Gln Leu Gln Gly Ile Val Ser
180 185 190
Ser Gly Tyr Gly Cys Ala Gln Lys Asn Lys Pro Gly Val Tyr Thr Lys
195 200 205
Val Cys Asn Tyr Val Asn Trp Ile Gln Gln Thr Ile Ala Ala Asn
210 215 220
<210> 125
<211> 223
<212> PRT
<213> 人工序列
<220>
<223> 猪胰蛋白酶变体125号
<400> 125
Ile Val Gly Gly Tyr Thr Cys Ala Ala Asn Ser Ile Pro Tyr Gln Val
1 5 10 15
Ser Leu Asn Ser Gly Ser His Asp Cys Gly Gly Ser Leu Ile Asn Ser
20 25 30
Gln Trp Val Val Ser Ala Ala His Cys Tyr Lys Ser Arg Ile Gln Val
35 40 45
Arg Leu Gly Glu His Asn Ile Asp Val Leu Glu Gly Asn Glu Gln Phe
50 55 60
Ile Asn Ala Ala Lys Ile Ile Thr His Pro Asn Phe Asn Gly Asn Thr
65 70 75 80
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Gly Thr Glu Cys Leu Ile Ser Gly Trp Gly Asn Thr Lys Ser Ser Gly
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Ile Cys Val Gly Phe Leu Glu Gly Gly Lys Asp Ala Cys Gln Gly Asp
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<210> 126
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<212> PRT
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<223> 猪胰蛋白酶变体126号
<400> 126
Ile Val Gly Gly Tyr Thr Cys Ala Ala Asn Ser Ile Pro Tyr Gln Val
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Ser Leu Asn Ser Gly Ser His Glu Cys Gly Gly Ser Leu Ile Asn Ser
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Gln Trp Val Val Ser Ala Ala His Cys Tyr Lys Ser Arg Ile Gln Val
35 40 45
Arg Leu Gly Glu His Asn Ile Asp Val Leu Glu Gly Asn Glu Gln Phe
50 55 60
Ile Asn Ala Ala Lys Ile Ile Thr His Pro Asn Phe Asn Gly Asn Thr
65 70 75 80
Leu Asp Asn Asp Ile Met Leu Ile Lys Leu Ser Ser Pro Ala Thr Leu
85 90 95
Asn Ser Arg Val Ala Thr Val Ser Leu Pro Arg Ser Cys Ala Ala Ala
100 105 110
Gly Thr Glu Cys Leu Ile Ser Gly Trp Gly Asn Thr Lys Ser Ser Gly
115 120 125
Ser Ser Tyr Pro Ser Leu Leu Gln Cys Leu Lys Ala Pro Val Leu Ser
130 135 140
Asp Ser Ser Cys Lys Ser Ser Tyr Pro Gly Gln Ile Thr Gly Asn Met
145 150 155 160
Ile Cys Val Gly Phe Leu Glu Gly Gly Lys Asp Ala Cys Gln Gly Asp
165 170 175
Ser Gly Gly Pro Val Val Cys Asn Gly Gln Leu Gln Gly Ile Val Ser
180 185 190
Ser Gly Tyr Gly Cys Ala Gln Lys Asn Lys Pro Gly Val Tyr Thr Lys
195 200 205
Val Cys Asn Tyr Val Asn Trp Ile Gln Gln Thr Ile Ala Ala Asn
210 215 220
<210> 127
<211> 223
<212> PRT
<213> 人工序列
<220>
<223> 猪胰蛋白酶变体127号
<400> 127
Ile Val Gly Gly Tyr Thr Cys Ala Ala Asn Ser Ile Pro Tyr Gln Val
1 5 10 15
Ser Leu Asn Ser Gly Ser His Arg Cys Gly Gly Ser Leu Ile Asn Ser
20 25 30
Gln Trp Val Val Ser Ala Ala His Cys Tyr Lys Tyr Arg Ile Gln Val
35 40 45
Arg Leu Gly Glu His Asn Ile Asp Val Leu Glu Gly Asn Glu Gln Phe
50 55 60
Ile Asn Ala Ala Lys Ile Ile Thr His Pro Asn Phe Asn Gly Asn Thr
65 70 75 80
Leu Asp Asn Asp Ile Met Leu Ile Lys Leu Ser Ser Pro Ala Thr Leu
85 90 95
Asn Ser Arg Val Ala Thr Val Ser Leu Pro Arg Ser Cys Ala Ala Ala
100 105 110
Gly Thr Glu Cys Leu Ile Ser Gly Trp Gly Asn Thr Lys Ser Ser Gly
115 120 125
Ser Ser Tyr Pro Ser Leu Leu Gln Cys Leu Lys Ala Pro Val Leu Ser
130 135 140
Asp Ser Ser Cys Lys Ser Ser Tyr Pro Gly Gln Ile Thr Gly Asn Met
145 150 155 160
Ile Cys Val Gly Phe Leu Glu Gly Gly Lys Asp Ala Cys Gln Gly Asp
165 170 175
Ser Gly Gly Pro Val Val Cys Asn Gly Gln Leu Gln Gly Ile Val Ser
180 185 190
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195 200 205
Val Cys Asn Tyr Val Asn Trp Ile Gln Gln Thr Ile Ala Ala Asn
210 215 220
<210> 128
<211> 223
<212> PRT
<213> 人工序列
<220>
<223> 猪胰蛋白酶变体128号
<400> 128
Ile Val Gly Gly Tyr Thr Cys Ala Ala Asn Ser Ile Pro Tyr Gln Val
1 5 10 15
Ser Leu Asn Ser Gly Ser His Arg Cys Gly Gly Ser Leu Ile Asn Ser
20 25 30
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35 40 45
Arg Leu Gly Glu His Asn Ile Asp Val Leu Glu Gly Asn Glu Gln Phe
50 55 60
Ile Asn Ala Ala Lys Ile Ile Thr His Pro Asn Phe Asn Gly Asn Thr
65 70 75 80
Leu Asp Asn Asp Ile Met Leu Ile Lys Leu Ser Ser Pro Ala Thr Leu
85 90 95
Asn Ser Arg Val Ala Thr Val Ser Leu Pro Arg Ser Cys Ala Ala Ala
100 105 110
Gly Thr Glu Cys Leu Ile Ser Gly Trp Gly Asn Thr Lys Ser Ser Gly
115 120 125
Ser Ser Pro Pro Ser Leu Leu Gln Cys Leu Lys Ala Pro Val Leu Ser
130 135 140
Asp Ser Ser Cys Lys Ser Ser Tyr Pro Gly Gln Ile Thr Gly Asn Met
145 150 155 160
Ile Cys Val Gly Phe Leu Glu Gly Gly Lys Asp Ala Cys Gln Gly Asp
165 170 175
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180 185 190
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195 200 205
Val Cys Asn Tyr Val Asn Trp Ile Gln Gln Thr Ile Ala Ala Asn
210 215 220
<210> 129
<211> 223
<212> PRT
<213> 人工序列
<220>
<223> 猪胰蛋白酶变体129号
<400> 129
Ile Val Gly Gly Tyr Thr Cys Ala Ala Asn Ser Ile Pro Tyr Gln Val
1 5 10 15
Ser Leu Asn Ser Gly Ser His Arg Cys Gly Gly Ser Leu Ile Asn Ser
20 25 30
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35 40 45
Arg Leu Gly Glu His Asn Ile Asp Val Leu Glu Gly Asn Glu Gln Phe
50 55 60
Ile Asn Ala Ala Lys Ile Ile Thr His Pro Asn Phe Asn Gly Asn Thr
65 70 75 80
Leu Asp Asn Asp Ile Met Leu Ile Lys Leu Ser Ser Pro Ala Thr Leu
85 90 95
Asn Ser Arg Val Ala Thr Val Ser Leu Pro Arg Ser Cys Ala Ala Ala
100 105 110
Gly Thr Glu Cys Leu Ile Ser Gly Trp Gly Asn Thr Lys Ser Ser Gly
115 120 125
Ser Ser Tyr Pro Ser Leu Leu Gln Cys Leu Lys Ala Pro Val Leu Ser
130 135 140
Asp Ser Ser Cys Lys Ser Ser Tyr Pro Gly Gln Ile Thr Gly Asn Met
145 150 155 160
Ile Cys Val Gly Phe Leu Glu Gly Gly Lys Asp Ala Cys Gln Gly Asp
165 170 175
Ser Gly Gly Pro Val Val Cys Asn Gly Gln Leu Gln Gly Ile Val Ser
180 185 190
Asn Gly Tyr Gly Cys Ala Gln Lys Asn Lys Pro Gly Val Tyr Thr Lys
195 200 205
Val Cys Asn Tyr Val Asn Trp Ile Gln Gln Thr Ile Ala Ala Asn
210 215 220
<210> 130
<211> 223
<212> PRT
<213> 人工序列
<220>
<223> 猪胰蛋白酶变体130号
<400> 130
Ile Val Gly Gly Tyr Thr Cys Ala Ala Asn Ser Ile Pro Tyr Gln Val
1 5 10 15
Ser Leu Asn Ser Gly Ser His Arg Cys Gly Gly Ser Leu Ile Asn Ser
20 25 30
Gln Trp Val Val Ser Ala Ala His Cys Tyr Lys Ser Arg Ile Gln Val
35 40 45
Arg Leu Gly Glu His Asn Ile Asp Val Leu Glu Gly Asn Glu Gln Phe
50 55 60
Ile Asn Ala Ala Lys Ile Ile Thr His Pro Asn Phe Asn Gly Asn Thr
65 70 75 80
Leu Asp Asn Asp Ile Met Leu Ile Lys Leu Ser Ser Pro Ala Thr Leu
85 90 95
Asn Ser Arg Val Ala Thr Val Ser Leu Pro Arg Ser Cys Ala Ala Ala
100 105 110
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115 120 125
Ser Ser Tyr Pro Ser Leu Leu Gln Cys Leu Lys Ala Pro Val Leu Ser
130 135 140
Asp Ser Ser Cys Lys Ser Ser Tyr Pro Gly Gln Ile Thr Gly Asn Met
145 150 155 160
Ile Cys Val Gly Phe Leu Glu Gly Gly Lys Asp Ala Cys Gln Gly Asp
165 170 175
Ser Gly Gly Pro Val Val Cys Asn Gly Gln Leu Gln Gly Ile Val Ser
180 185 190
Cys Gly Tyr Gly Cys Ala Gln Lys Asn Lys Pro Gly Val Tyr Thr Lys
195 200 205
Val Cys Asn Tyr Val Asn Trp Ile Gln Gln Thr Ile Ala Ala Asn
210 215 220

Claims (16)

1.一种猪胰蛋白酶变体，其包含
与SEQ ID NO:1具有至少90％序列同一性的氨基酸序列或者由所述氨基酸序列组成，
其中所述氨基酸序列与SEQ ID NO:1相比不同之处至少在于在对应于根据SEQ ID NO:1的天然猪胰蛋白酶的F24、S44、D56、G78、Y131、S172和W193的一个或多个位置处的一个或多个氨基酸取代，
前提是所述氨基酸序列不是根据SEQ ID NO:1的天然猪胰蛋白酶；并且
前提是所述氨基酸序列不是根据SEQ ID NO:2的猪变体胰蛋白酶S172A。
2.根据权利要求1所述的猪胰蛋白酶变体，
其中对应于F24的位置处的氨基酸被选自以下的氨基酸取代：Ala、Asn、Arg、Gln、Ile、Leu、Lys、Met、Ser、Thr和Val；
其中对应于S44的位置处的氨基酸被选自以下的氨基酸取代：Leu和Pro；
其中对应于D56的位置处的氨基酸被选自以下的氨基酸取代：Ala、Asn、His和Trp；
其中对应于G78的位置处的氨基酸被选自以下的氨基酸取代：Ala、Glu、Pro、Ser和Tyr；
其中对应于Y131的位置处的氨基酸被选自以下的氨基酸取代：Ala、Asn、Asp、Cys、Gln、Glu、Gly、His、Ile、Leu、Met、Ser、Thr、Trp、Val；
其中对应于S172的位置处的氨基酸被选自以下的氨基酸取代：Ala、Cys和Thr；并且/或者
其中对应于W193的位置处的氨基酸被选自以下的氨基酸取代：Phe、Ser、Thr和Tyr。
3.根据权利要求1至2中任一项所述的猪胰蛋白酶变体，其中所述氨基酸序列与SEQ IDNO:1相比另外的不同之处至少在于在对应于根据SEQ ID NO:1的天然猪胰蛋白酶的R99、R107、K125和K170的一个或多个位置处的一个或多个氨基酸取代。
4.根据权利要求3所述的猪胰蛋白酶变体，
其中对应于R99的位置处的氨基酸被选自以下的氨基酸取代：Ala、Asn、Asp、Glu、Gly、His、Leu、Phe、Thr、Trp和Tyr；
其中对应于R107的位置处的氨基酸被选自以下的氨基酸取代：Asp、Gly、Pro、Ser和Thr；
其中对应于K125的位置处的氨基酸被选自以下的氨基酸取代：Ala、Cys、Gln、Glu、Gly、His、Leu、Ser和Tyr；并且/或者
其中对应于K170的位置处的氨基酸被选自以下的氨基酸取代：Ala、Asn、Gly和Tyr。
5.根据权利要求1至4中任一项所述的猪胰蛋白酶变体，其中所述氨基酸序列是SEQ IDNO:3，
其中
-Xaa24是选自以下的氨基酸：Ala、Asn、Arg、Gln、Ile、Leu、Lys、Met、Phe、Ser、Thr和Val；
-Xaa44是选自以下的氨基酸：Leu、Pro和Ser；
-Xaa56是选自以下的氨基酸：Ala、Asn、Asp、His和Trp；
-Xaa78是选自以下的氨基酸：Ala、Glu、Gly、Pro、Ser和Tyr；
-Xaa99是选自以下的氨基酸：Ala、Arg、Asn、Asp、Glu、Gly、His、Leu、Phe、Thr、Trp和Tyr；
-Xaa107是选自以下的氨基酸：Arg、Asp、Gly、Pro、Ser和Thr；
-Xaa125是选自以下的氨基酸：Ala、Cys、Gln、Glu、Gly、His、Leu、Lys、Ser和Tyr；
-Xaa131是选自以下的氨基酸：Ala、Asn、Asp、Cys、Gln、Glu、Gly、His、Ile、Leu、Met、Ser、Thr、Trp、Tyr和Val；
-Xaa170是选自以下的氨基酸：Ala、Asn、Gly、Lys和Tyr；
-Xaa172是选自以下的氨基酸：Ala、Cys、Ser和Thr；并且/或者
-Xaa193是选自以下的氨基酸：Phe、Ser、Thr、Trp和Tyr；
前提是SEQ ID NO:3不是根据SEQ ID NO:1的猪野生型胰蛋白酶；并且
前提是SEQ ID NO:3不是根据SEQ ID NO:2的猪变体胰蛋白酶S172A。
6.根据权利要求1至5中任一项所述的猪胰蛋白酶变体，其中所述氨基酸序列选自SEQID NO:4、SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:9、SEQ IDNO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:13、SEQ ID NO:14、SEQ ID NO:15、SEQ IDNO:16、SEQ ID NO:17、SEQ ID NO:18、SEQ ID NO:19、SEQ ID NO:20、SEQ ID NO:21、SEQ IDNO:22、SEQ ID NO:23、SEQ ID NO:24、SEQ ID NO:25、SEQ ID NO:26、SEQ ID NO:27、SEQ IDNO:28、SEQ ID NO:29、SEQ ID NO:30、SEQ ID NO:31、SEQ ID NO:32、SEQ ID NO:33、SEQ IDNO:34、SEQ ID NO:35、SEQ ID NO:36、SEQ ID NO:37、SEQ ID NO:38、SEQ ID NO:39、SEQ IDNO:40、SEQ ID NO:41、SEQ ID NO:42、SEQ ID NO:43、SEQ ID NO:44、SEQ ID NO:45、SEQ IDNO:46、SEQ ID NO:47、SEQ ID NO:48、SEQ ID NO:49、SEQ ID NO:50、SEQ ID NO:51、SEQ IDNO:52、SEQ ID NO:53、SEQ ID NO:54、SEQ ID NO:55、SEQ ID NO:56、SEQ ID NO:57、SEQ IDNO:58、SEQ ID NO:59、SEQ ID NO:60、SEQ ID NO:61、SEQ ID NO:62、SEQ ID NO:63、SEQ IDNO:64、SEQ ID NO:65、SEQ ID NO:66、SEQ ID NO:67、SEQ ID NO:68、SEQ ID NO:69、SEQ IDNO:70、SEQ ID NO:71、SEQ ID NO:72、SEQ ID NO:73、SEQ ID NO:74、SEQ ID NO:75、SEQ IDNO:76、SEQ ID NO:77、SEQ ID NO:78、SEQ ID NO:79、SEQ ID NO:80、SEQ ID NO:81、SEQ IDNO:82、SEQ ID NO:83、SEQ ID NO:84、SEQ ID NO:85、SEQ ID NO:86、SEQ ID NO:87、SEQ IDNO:88、SEQ ID NO:89、SEQ ID NO:90、SEQ ID NO:91、SEQ ID NO:92、SEQ ID NO:93、SEQ IDNO:94、SEQ ID NO:95、SEQ ID NO:96、SEQ ID NO:97、SEQ ID NO:98、SEQ ID NO:99、SEQ IDNO:100、SEQ ID NO:101、SEQ ID NO:102、SEQ ID NO:103、SEQ ID NO:104、SEQ ID NO:105、SEQ ID NO:106、SEQ ID NO:107、SEQ ID NO:108、SEQ ID NO:109、SEQ ID NO:110、SEQ IDNO:111、SEQ ID NO:112、SEQ ID NO:113、SEQ ID NO:114、SEQ ID NO:115、SEQ ID NO:116、SEQ ID NO:117、SEQ ID NO:118和SEQ ID NO:119。
7.根据权利要求1至6中任一项所述的猪胰蛋白酶变体，其中
(i)所述变体能够切割具有通式A-Lys-Thr-Arg-Arg-B的肽，以产生通式A-Lys-Thr-Arg-Arg的切割产物，产率为至少80％，
其中A是由一个或多个氨基酸组成的氨基酸序列；并且
其中B是由一个或多个氨基酸组成的氨基酸序列；
并且/或者
(ii)与根据SEQ ID NO:2的猪变体胰蛋白酶S172A相比，所述变体表现出在位置B32-Arg的C末端的位置处对人前胰岛素原、甘精前胰岛素原、赖脯前胰岛素原、门冬前胰岛素原或赖谷前胰岛素原的切割的增加的选择性。
8.一种核酸分子，其编码
-根据权利要求1至7中任一项所述的猪胰蛋白酶变体，或者
-根据权利要求1至7中任一项所述的猪胰蛋白酶变体的胰蛋白酶原前体分子，
其中所述核酸分子任选地包含在载体中或任选地整合到基因组中。
9.一种宿主细胞，其包含根据权利要求8所述的核酸分子。
10.一种产生根据权利要求1至7中任一项所述的猪胰蛋白酶变体的方法，其包括以下步骤：
培养根据权利要求9所述的宿主细胞；以及
从培养基或从所述宿主细胞分离所述猪胰蛋白酶变体的胰蛋白酶原前体分子；以及
任选地活化所述胰蛋白酶原前体分子，从而获得所述猪胰蛋白酶变体。
11.根据权利要求1至7中任一项所述的猪胰蛋白酶变体或根据权利要求8所述的核酸分子或根据权利要求9所述的宿主细胞，其用于在制造食品成分或饲料成分的工艺内使用。
12.根据权利要求1至7中任一项所述的猪胰蛋白酶变体在用于产生人胰岛素、胰岛素类似物或胰岛素衍生物的方法中的用途。
13.根据权利要求12所述的用途，其中所述胰岛素类似物选自门冬胰岛素、赖脯胰岛素、赖谷胰岛素和甘精胰岛素。
14.根据权利要求1至7中任一项所述的猪胰蛋白酶变体用于切割具有通式A-Lys-Thr-Arg-Arg-B的蛋白质或肽的用途，
其中A是由一个或多个氨基酸组成的氨基酸序列；并且
其中B是由一个或多个氨基酸组成的氨基酸序列。
15.根据权利要求14所述的用途，其中所述切割产生切割产物A-Lys-Thr-Arg-Arg。
16.根据权利要求15所述的用途，其中所述切割产生所述切割产物A-Lys-Thr-Arg-Arg，产率为至少80。
CN201980026139.4A 2018-02-22 2019-02-22 猪胰蛋白酶变体 Pending CN111989401A (zh)

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