CN1183809A - 具有降低吸附作用提高水解作用的蛋白酶k变异体 - Google Patents
具有降低吸附作用提高水解作用的蛋白酶k变异体 Download PDFInfo
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- CN1183809A CN1183809A CN96193792A CN96193792A CN1183809A CN 1183809 A CN1183809 A CN 1183809A CN 96193792 A CN96193792 A CN 96193792A CN 96193792 A CN96193792 A CN 96193792A CN 1183809 A CN1183809 A CN 1183809A
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Abstract
本发明涉及蛋白酶K的变异体,它们对野生型蛋白酶K的氨基酸序列进行了改良,野生型氨基酸序列包含第一环区、第二环区、第三环区、第四环区、第五环区和第六环区;其中改良的氨基酸序列是在一个或多个环区的特定位置上含有与野生型蛋白酶K不同的氨基酸(即替换),因此,与野生型蛋白酶K相比,蛋白酶K变异体降低了对不溶底物的吸附作用,并提高了对不溶底物的水解作用。本发明也涉及编码这些蛋白酶K变异体的DNA序列。本发明还涉及包含这些蛋白酶K变异体的用于清洁多种表面的组合物。
Description
技术领域
本发明涉及用于各种洗涤剂的新的酶变异体及编码这些酶变异体的DNA序列。
发明背景
在天然存在的蛋白质中，酶组成了其中最大的一类。每类酶通常催化(加速一个反应而本身不被消耗)不同类型的化学反应。其中一类酶如蛋白酶，已知它们具有水解(将一个化合物断裂成两个或更多的简单化合物，并在断裂的化学键的任何一端吸收水分子的H和OH部分)其它蛋白质的能力。这种水解蛋白酶的能力已通过在洗衣用洗涤剂制品中加入天然存在的或蛋白工程改造的蛋白酶作为添加剂而得到利用。衣服上许多污迹都是蛋白质性的，而特异性宽的蛋白酶能基本上增强去除这些污迹的能力。
遗憾的是，这些蛋白质在它们天然的细菌环境中的效力水平常常不能转移到相对非天然的洗涤环境中去。尤其是，当不在这些酶的天然环境中使用时，蛋白酶的一些特征，如热稳定性、pH稳定性、氧化稳定性和底物特异性不是所需的最佳状态。
蛋白酶的氨基酸序列决定了蛋白酶的性质。蛋白酶中氨基酸序列的改变，根据改变氨基酸序列的位置、性质和/或数量，可不同程度地改变酶的性质，甚至可使酶灭活。已采用了几种方法改变野生型蛋白酶的氨基酸序列以试图改良它们的性质，达到在洗涤环境中提高蛋白酶效力的目的。这些方法包括改变氨基酸序列以增强热稳定性并提高在不同环境下的氧化稳定性。
尽管本领域中已描述了多种方法，但仍需继续寻找新的有效的蛋白酶的变异体，用于清洁多种表面。
发明目的
本发明的一个目的是提供相对于野生型蛋白酶K具有更强的水解作用的蛋白酶K酶变异体。
本发明的另一目的是提供含有这些枯草杆菌蛋白酶变异体的洗涤剂。
发明概述
本发明涉及蛋白酶K的变异体，它们对野生型蛋白酶K的氨基酸序列中某些序列进行改良，野生型氨基酸序列包括第一环区、第二环区、第三环区、第四环区和第五环区和第六环区；其中改良的氨基酸序列是在一个或多个环区的特定位置上含有与野生型蛋白酶K不同的氨基酸(即替换)，因此，与野生型蛋白酶K相比，蛋白酶K变异体降低了对不溶底物的吸附作用，并提高了对不溶底物的水解作用。本发明也涉及编码这些蛋白酶K变异体的DNA序列。本发明还涉及包含有这些蛋白酶K变异体的用于清洁多种表面的组合物。
发明描述I.蛋白酶K变异体
本发明涉及枯草杆菌蛋白酶，尤其是蛋白酶K，通过使编码这些酶的各种核苷酸序列发生突变从而改变该酶的氨基酸序列来改良这些酶。本发明改良的枯草杆菌蛋白酶(下文称为“蛋白酶K变异体”)与野生型枯草杆菌蛋白酶相比，降低了对不溶底物的吸附作用并提高了对不溶底物的水解作用。本发明还涉及编码这些蛋白酶K变异体的DNA序列。
本发明中的枯草杆菌蛋白酶属于已知为蛋白酶的一类酶。蛋白酶是一种断裂肽键的催化剂。蛋白酶中的一类是丝氨酸蛋白酶，丝氨酸蛋白酶的特征是在其活性中心有一个必需的丝氨酸残基。
有资料证实了酶对可溶底物的水解速率随酶浓度的增大而提高这种观测结果。对表面结合的底物也似乎是这样，例如在许多洗涤应用中遇到的，水解速率随表面浓度的增大而提高，这已为事实所证实。(Brode，P.F.III和D.S.Rauch，LANGMUIR，“枯草杆菌蛋白酶BPN：对固定化底物的活性(Subtilisin BPN：Activity on an ImmobilizedSubstrate)”，1992年第8卷1325-1329页)。事实上，已发现当酶的表面浓度发生变化时，对不溶底物的水解速率与表面浓度呈线性依赖关系。(Rubingh，D.N.和M D.Bauer，“蛋白酶在蛋白-溶液界面的水解催化作用(Catalysis of Hydrolysis by Proteases at theProtein-Solution Interface)”见I.Noda和D.N.Rubingh编，Elsevier1992年出版“聚合物溶液，混合物和界面(POLYMER SOLUTIONS，BLENDS AND INTERFACES)”第464页)。令人吃惊的是，当我们试图利用这一原则寻找具有更好的洗涤性能的变异的蛋白酶时，我们找不到吸附越好性能越好的酶。实际上，我们意外地测定到了相反的情况：降低酶对底物的吸附作用将提高底物的水解作用(即更好的洗涤性能)。
虽然不希望拘泥于理论，但可以认为当变异体相互比较时，性能的提高是源于这样一个事实，即吸附作用小的酶结合力也小，从而在表面上有更高的移动性，将不溶的蛋白质底物从表面上除去。在可比较的酶溶液浓度下，这种提高的移动性足以胜过在表面施加更高浓度的酶所带来的好处。
本文所述的突变被指定为改变(即降低)酶对表面结合的污垢的吸附作用。在蛋白酶K中，某些氨基酸在酶分子上形成外环。为了讨论的方便，这些环被称作第一、第二、第三、第四和第五环区。具体地说，64-71位形成第一环区；95-107位形成第二环区；133-140位形成第三环区；160-170位形成第四环区；190-194位形成第五环区；203-223位形成第六环区(位置序号类似于野生型枯草杆菌蛋白酶K中氨基酸序列的位置(SEQ ID NO.1)。
据信这些环区在酶分子对表面结合的肽的吸附作用中起着重要作用，一个或更多的环区上特定的突变将显著影响这种吸附作用。虽然不希望受理论的限制，但至少有两个原因可使人相信这些环区对蛋白酶K分子的吸附作用是非常重要的。第一，组成环区的氨基酸可与酶分子作用的表面紧密接触。第二，环区与蛋白酶K分子上活性中心和结合口袋的邻近使其在酶对表面结合的底物(肽/蛋白质污垢)的催化产生的吸附作用中起着作用。
本文中，“变异体”指氨基酸序列与野生型酶不同的酶。
本文中，“突变的蛋白酶K DNA”指编码蛋白酶K变异体的DNA序列。
本文中，“野生型蛋白酶K”指SEQ ID NO.1所代表的酶。Gunkel，F.A.和Gassen H.G.对蛋白酶K的氨基酸序列有更多的描述，见“来自Tritirachium album Limber的蛋白酶K：染色体基因的鉴定及其cDNA在大肠杆菌中的表达(Proteinase K from Tritirachium albumLimber：Characterization of the Chromosomal Gene andExpressim of the cDNA in Escherichia coli)”欧洲生物化学杂志(EUR.J.BIOCHEM.)1989年179卷185-194页，在此引入作为参考。
本文中，术语“蛋白酶K野生型氨基酸序列”包括SEQ ID NO：1以及除64-71，95-107，133-140，160-170，190-194和203-223位外有修饰氨基酸序列的SEQ ID NO：1。
本文中，“更亲水的氨基酸”指比参照下面的亲水性表的对照氨基酸具有更高亲水性的其它任何氨基酸。下面的亲水性表(表1)按亲水性升高由上向下顺序排列出了氨基酸(见Hopp，T.P.，和Woods，K.R.，“由氨基酸序列推测蛋白质的抗原决定簇(Prediction of ProteinAntigenic Determinants from Amino Acid Sequences)”，美国国家科学院学报(PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCEUSA)1981年78卷3824-3828页，在此引入作为参考)。
表1
氨基酸 亲水性数值
Trp -3.4
Phe -2.5
Tyr -2.3
Leu，Ile -1.8
Val -1.5
Met -1.3
Cys -1.0
Ala，His -0.5
Thr -0.4
Pro，Gly -0.0
Gln，Asn 0.2
Ser 0.3
Arg+，Lys+，Glu-，Asp- 3.0
表1也指明了哪些氨基酸带有电荷(该特征是基于pH为大约8-9)。带正电荷的氨基酸是Arg和Lys，带负电荷的氨基酸是Glu和Asp，剩下的氨基酸是中性的。在本发明的-个优选实施方案中，替代的氨基酸为中性或带负电荷的，更优选带负电荷的(即Glu或Asp)。
因此，例如，“用中性或带负电荷的亲水性相同或更强的氨基酸替换Gln”是指Gln将被Asn(与Gln有相同亲水性)或Ser、Glu或Asp(它们均比Gln亲水性强)替换；与Gln相比，它们均是中性的或带负电荷的并都具有更高的亲水性值。同样，“用中性或带负电荷的亲水性更强的氨基酸替换Pro”是指Pro将被Gln、Asn、Ser、Glu或Asp替换。
在本发明的一个实施方案中，蛋白酶K变异体是对蛋白酶K野生型氨基酸序列进行改良，其中在野生型氨基酸序列的第一环区、第二环区、第三环区、第四环区、第五环区或第六环区中的一个或多个环区中的一个或多个位置包含替换；因此蛋白酶K变异体与野生型蛋白酶K相比，降低了对不溶底物的吸附作用，而提高了对不溶底物的水解作用。
在本发明的一个优选实施方案中，一个或多个环区中一个或多个位置的替换氨基酸(参照表1)是中性或带负电荷的，并且与野生型氨基酸序列中被替换位置的氨基酸相比，具有相同或更强的亲水性，优选具有更强亲水性的氨基酸。A.在第一环区中的替换
当替换发生在第一环区时，替换发生在64、65、66、67、68、70或71位中的一个或多个位置。
当替换发生在64位时，替代氨基酸是Asp或Glu。
当替换发生在65位时，替代氨基酸是Glu。
当替换发生在66位时，替代氨基酸是Asn、Asp、Gln、Glu、Pro或Ser。
当替换发生在67位时，替代氨基酸是Asp、Gln、Glu或Ser。
当替换发生在68位时，替代氨基酸是Asn、Asp、Gln、Glu、Pro或Ser。
当替换发生在70位时，替代氨基酸是Asn、Asp、Gln、Glu、Pro或Ser。
当替换发生在71位时，替代氨基酸是Asn、Asp、Gln、Glu、Gly、Pro或Ser。B.在第二环区中的替换
当替换发生在第二环区时，替换发生在95、96、97、98、99、100、101、102、103、104、105、106或107位中的一个或多个位置。
当替换发生在95位时，替代氨基酸是Ala、Asn、Asp、Cys、Gln、Glu、Gly、His、Met、Pro、Ser或Thr。
当替换发生在96位时，替代氨基酸是Ala、Asn、Asp、Cys、Gln、Glu、Gly、His、Ile、Met、Pro、Ser、Thr或Val。
当替换发生在97位时，替代氨基酸是Glu。
当替换发生在98位时，替代氨基酸是Glu。
当替换发生在99位时，替代氨基酸是Asp、Gln、Glu或Ser。
当替换发生在100位时，替代氨基酸是Asn、Asp、Gln、Glu、Pro或Ser。
当替换发生在101位时，替代氨基酸是Asp或Glu。
当替换发生在102位时，替代氨基酸是Asn、Asp、Gln、Glu、Pro或Ser。
当替换发生在103位时，替代氨基酸是Asn、Asp、Glu或Ser。
当替换发生在104位时，替代氨基酸是Ala、Asn、Asp、Cys、Gln、Glu、Gly、His、Ile、Leu、Met、Pro、Ser、Thr或Val。
当替换发生在105位时，替代氨基酸是Asp或Glu。
当替换发生在106位时，替代氨基酸是Asn、Asp、Gln、Glu、Gly、Pro或Ser。
当替换发生在107位时，替代氨基酸是Ala、Asn、Asp、Cys、Gln、Glu、Gly、His、Leu、Met、Pro、Ser、Thr或Val。C.在第三环区中的替换
当替换发生在第三环区时，替代发生在133、134、135、136、137、138、139或140位中的一个或多个位置。其中
当替换发生在133位时，替代氨基酸是Ala、Asn、Asp、Cys、Gln、Glu、Gly、His、Ile、Met、Pro、Ser、Thr或Val。
当替换发生在134位时，替代氨基酸是Asn、Asp、Gln、Glu、Pro或Ser。
当替换发生在135位时，替代氨基酸是Asn、Asp、Gln、Glu、Pro或Ser。
当替换发生在136位时，替代氨基酸是Asn、Asp、Gln、Glu、Pro或Ser。
当替换发生在137位时，替代氨基酸是Ala、Asn、Asp、Cys、Gln、Glu、Gly、His、Ile、Leu、Met、Pro、Ser、Thr或Val。
当替换发生在138位时，替代氨基酸是Asp或Glu。
当替换发生在139位时，替代氨基酸是Asp或Glu。和
当替换发生在140位时，替代氨基酸是Asp或Glu。D.在第四环区中的替换
当替换发生在第四环区时，替换发生在160、161、162、163、164、165、166、167、168、169或170位中的一个或多个位置。其中
当替换发生在160位时，替换氨基酸是Asn、Asp、Gln、Glu、Pro或Ser。
当替换发生在161位时，替换氨基酸是Asp、Gln、Glu或Ser。
当替换发生在162位时，替换氨基酸是Asp、Gln、Glu或Ser。
当替换发生在163位时，替换氨基酸是Asp、Gln、Glu或Ser。
当替换发生在164位时，替代氨基酸是Asn、Asp、Gln、Glu、Gly、His、Pro、Ser或Thr。
当替换发生在165位时，替代氨基酸是Glu。
当替换发生在166位时，替代氨基酸是Asn、Asp、Gln、Glu、Gly、His、Pro、Ser或Thr。
当替换发生在167位时，替代氨基酸是Asp或Glu。
当替换发生在168位时，替代氨基酸是Asp、Gln、Glu或Ser。
当替换发生在169位时，替代氨基酸是Ala、Asn、Asp、Cys、Gln、Glu、Gly、His、Ile、Leu、Met、Pro、Ser、Thr或Val。和
当替换发生在170位时，替代氨基酸是Asp或Glu。E.在第五环区中的替换
当替换发生在第五环区时，替换发生在190、191、192、193或194位中的一个或多个位置。其中
当替换发生在190位时，替代氨基酸是Asp或Glu。
当替换发生在191位时，替代氨基酸是Asp或Glu。
当替换发生在192位时，替代氨基酸是Ala、Asn、Asp、Cys、Gln、Glu、Gly、His、Ile、Leu、Met、Pro、Ser、Thr、Tyr或Val。
当替换发生在193位时，替代氨基酸是Asp或Glu。和
当替换发生在194位时，替代氨基酸是Asp、Gln、Glu或Ser。F.在第六环区中的替换
当替换发生在第六环区时，替换发生在203、204、205、206、207、208、209、210、211、212、213、214、215、216、217、218、219、220、221、222或223位中的一个或多个位置。其中
当替换发生在203位时，替代氨基酸是Asn、Asp、Gln、Glu、Pro或Ser。
当替换发生在204位时，替代氨基酸是Asn、Asp、Gln、Glu、Gly或Ser。
当替换发生在205位时，替代氨基酸是Asn、Asp、Gln、Glu、Pro或Ser。
当替换发生在206位时，替代氨基酸是Asn、Asp、Gln、Glu、Gly、Pro或Ser。
当替换发生在207位时，替代氨基酸是Asp或Glu。
当替换发生在208位时，替代氨基酸是Ala、Asn、Asp、Cys、Gln、Glu、Gly、His、Leu、Met、Pro、Ser、Thr或Val。
当替换发生在209位时，替代氨基酸是Ala、Asn、Asp、Cys、Gln、Glu、Gly、His、Ile、Met、Pro、Ser、Thr或Val。
当替换发生在210位时，替代氨基酸是Asp或Glu。
当替换发生在211位时，替代氨基酸是Asn、Asp、Gln、Glu、Gly、Pro或Ser。
当替换发生在212位时，替代氨基酸是Ala、Asn、Asp、Cys、Gln、Glu、Gly、His、Ile、Leu、Met、Phe、Pro、Ser、Thr、Tyr或Val。
当替换发生在213位时，替代氨基酸是Ala、Asn、Asp、Cys、Gln、Glu、Gly、His、Leu、Met、Pro、Ser、Thr或Val。
当替换发生在214位时，替代氨基酸是Asn、Asp、Gln、Glu、Pro或Ser。
当替换发生在215位时，替代氨基酸是Asn、Asp、Gln、Glu、Pro或Ser。
当替换发生在216位时，替代氨基酸是Asp或Glu。
当替换发生在217位时，替代氨基酸是Asn、Asp、Gln、Glu、Gly、Pro或Ser。
当替换发生在218位时，替代氨基酸是Asp或Glu。
当替换发生在219位时，替代氨基酸是Asp或Glu。
当替换发生在220位时，替代氨基酸是Ala、Asn、Asp、Cys、Gln、Glu、Gly、His、Leu、Met、Pro、Ser、Thr或Val。
当替换发生在221位时，替代氨基酸是Asp或Glu。
当替换发生在222位时，替代氨基酸是Asn、Asp、Gln、Glu、Pro或Ser。
当替换发生在223位时，替代氨基酸是Asn、Asp、Gln、Glu、Pro或Ser。G.酶变异体的制备
实施例1
突变型蛋白酶K DNA
构建一个含野生型蛋白酶K基因的噬菌粒(“PKP”)。质粒pUC119(Vieira.J和Messing，J.“单链质粒DNA的制备(Production ofSingle-Stranded Plasmid DNA)”，酶学方法(METHODS INENZYMOLOGY)1989年153卷3-11页)中2.8Kbp PvuII限制酶片段克隆到质粒pUB110(Bacillus Genetic Stock Center，Columbus，OH 1E9)的PvuII位点上。pUC119-pUB110杂交质粒被命名为pJMA 601。将解淀粉芽孢杆菌枯草杆菌蛋白酶基因克隆到质粒pJMA 601中。枯草杆菌蛋白酶基因被修饰后含有两个BamHI位点。一个BamHI位点在编码Gly和Lys的DNA之间，即前区中第二和第三个氨基酸残基的DNA之间。另一个BamHI位点位于TAA终止序列之后。利用反转录和聚合酶链反应从RNA中扩增出蛋白酶K的cDNA，其中寡核苷酸引物上除含与蛋白酶K cDNA相同的序列外，还各含一个BamHI位点。被扩增的片段包含从蛋白酶K前区起始处Ala到成熟蛋白酶C末端Ala间的DNA序列。扩增的片段用于替换解淀粉芽孢杆菌枯草杆菌蛋白酶基因中两个BamHI位点间的DNA序列。将噬菌粒PKP转化到大肠杆菌Ung-菌株CJ 236中并用VCSM 13辅助噬菌体产生含尿嘧啶的单链DNA模板(Kunkel，T.A.，J.D.Roberts和R.A.Zakour，“快速有效无需表型选择的位点专一诱变(Rapid and efficient site-specificmutagenesis without phenotypic selection)”，酶学方法(METHODS INENZYMOLOGY)，1987年154卷367-382页；改良的方法见Yuckenberg，P.D.，F.Witney，J.Geisselsoder和J.McClary“利用含尿嘧啶的DNA和噬菌粒载体的体外定点诱变(Site-directed in vitro mutagenesisusing urail-containing DNA and phagemid vectors)”，M.J.McPherson编1991年版“定向诱变实用方法(DIRECTED MUTAGENESIS-A PRACTICAL APPROACH)”27-48页，两者均引于此以作参考)。用Zoller和Smith的单引物定点诱变修饰方法(Zoller，M.J.和M.Smith，“用M13衍生载体进行寡核苷酸指导的诱变：一种在任何DNA片段上产生点突变的有效而常用的方法(0ligonucleotide-directedmutagenesis using M13-derived vectors：an efficient andgeneral procedure for the production of point mutations inany fragment of DNA)”，核酸研究(NUCLEIC ACIDS RESEARCH)，1982年10卷6487-6500页，在此引入作为参考)产生所有的突变体(基本上如以上Yuckenberg等(1991)介绍的)。寡核苷酸用AppliedBiosystem Inc.公司380B DNA合成仪合成。诱变反应产物转化到大肠杆菌MM 294菌株(American Type Culture Collection E.coli.33625)中。所有突变体均由DNA测序所证实且分离的DNA被转化到枯草芽胞杆菌表达型菌株BG 2036中(Yang，M.Y.，E.Ferrari和D.J.Henner，“枯草芽胞杆菌中性蛋白酶基因的克隆以及用克隆基因产生体外诱导的缺失突变(Cloning of the Neutral Protease Gene ofBacillus subtilis and the Use of the Cloned Gene to Createan In Vitro-derived Deletion Mutation)”，细菌学杂志(JOURNAL OF BACTERIOLOGY)1984年160卷15-21页)。对于一些环区突变体，使用在相应环区含终止密码子移码突变的修饰PKP产生尿嘧啶模板。寡核苷酸被设计成可恢复正确的阅读框并编码64，65，66，67，68，70，71，95，96，97，98，99，100，101，102，103，104，105，106，107，133，134，135，136，137，138，139，140，160，161，162，163，164，165，166，167，168，169，170，190，191，192，193，194，203，204，205，206，207，208，209，210，211，212，213，214，215，216，217，218，219，220，221，222或223位的随机替换(这些密码子中所有三种碱基用等摩尔和/或比例不同的所有四种核苷酸的混合物)。修正了移码终止并产生有功能酶的突变通过它们消化酪蛋白的能力得到鉴别。随机替换由DNA测序确定。
实施例2
发酵
含有研究中的枯草杆菌蛋白酶突变体的枯草芽胞杆菌细胞(BG2036)在1升LB-葡萄糖肉汤培养基中长至对数中期后，接种至BiostatED发酵罐(B.Braun Biotech，Inc.，Allentown，Pennsylvania)中，体积为10升。发酵培养基中含酵母抽提物、淀粉、抗泡沫剂、缓冲剂和痕量的矿质(见“发酵实用方法(FERMENTATION：A PRACTICALAPPROACH)”，B.McNeil和L.M.Harvey编，1990年版)。在发酵过程中肉汤培养基pH保持在7.0。加入氯霉素以对诱变质粒进行抗生素选择。细胞在37℃生长过夜至A600为约60，然后收获。
实施例3
纯化
通过下面的步骤取用发酵肉汤获得纯酶。通过离心从肉汤培养基中除去枯草杆菌细胞，然后用100K的截止滤膜除去细小的颗粒使肉汤澄清。接着用10K的截止滤膜浓缩并通过流动透析降低离子强度，并用0.025M MES缓冲剂(2-(N-吗啉代)乙磺酸)调pH至5.5。酶上样于阳离子交换色谱柱或亲和吸附色谱柱进一步纯化，用NaCl或丙二醇梯度液从柱中洗脱该酶(见Scopes.R.K.“蛋白纯化原理与方法(PROTEIN PURIFICATION PRINCIPLES AND PRACTICE)”，在此引入作为参考)。
pNA实验(DelMar，E.G.，C.Largman，J.W.Brodrick和M.C.Geokas，分析生物化学(ANAL.BIOCHEM.)1979年99卷316-320页，在此引入作为参考)用来检测梯度洗脱时分部收集液中的活性酶浓度。该实验测量了当酶水解可溶性合成底物琥珀酰-丙氨酸-丙氨酸-脯氨酸-苯丙氨酸-对硝基苯胺(sAAPF-pNA)时，对硝基苯胺的释放速率。用分光光度计于410nm处测量水解反应中黄色的生成速率，其与活性酶的浓度成比例。另外，280nm处测定的吸光值用来确定总蛋白浓度。活性酶/总蛋白的比值表示酶纯度，并用来确定将合并为贮存液的各分部收集液。
为了避免贮存中的酶自水解，向合并的从色谱柱中得到的分部收集液中加入等重的丙二醇。纯化完成后，贮存酶溶液的纯度用SDS-PAGE(十二烷基硫酸钠聚丙烯酰胺凝胶电泳)检测，绝对酶浓度用购自Sigma化学公司(St.Louis，Missouri)的胰蛋白酶抑制剂II-T型：火鸡卵清，通过活性位点滴定法确定。测量所得的转换因数将告诉我们在酶分子中不同位置的哪些改变将产生比野生型酶对可溶底物pNA具有更高活性的酶变异体。
使用之前，酶贮存液用Sephadex G 25(Pharmacia，Piscataway，NewJersey)分子排阻过滤柱洗脱除去丙二醇并更换缓冲剂。酶贮存液中MES缓冲剂被换成含0.01M CaCl2并用HCl调pH至8.6的0.1M Tris缓冲剂(Tris(羟甲基氨基甲烷))。所有实验在25℃，pH8.6 Tris缓冲剂中进行。H.酶变异体的鉴定
实施例4
模型表面的制备
购自CPG公司的氨丙基人工微孔玻璃(CPG)用作支持物，共价附着购自Bachem公司(Torrence，California)的sAAPF-pNA底物。反应在二甲亚砜中进行，(1-乙基-3-[3-(二甲基氨基)丙基]碳二亚胺盐酸盐)(EDC)用作偶合剂。反应完后(用pNA实验检测)，除去过量的溶剂，CPG：sAAPF-pNA用二甲亚砜(DMSO)和双蒸水淋洗，随后在干燥箱中约70℃用N2吹干。反应方案和固定化底物的制备按Brode，P.F.III，和D.S.Rauch所述进行，见“枯草杆菌蛋白酶BPN：对固定化底物的活性(Subtilisin BPN：Activity on anImmobilized Substrate)”，LANGMUIR，1992年8卷1325-1329页，在此引入作为参考。
CPG表面具有62,000±7,000 pNA分子/μm2。其表面积与CPG公司为我们所购的CPG提供的数据50.0m2/g相比没有改变。这表明往CPG上加sAAPF-pNA的方法没有损坏其多孔的结构(平均直径486)。
实施例5
表面水解实验
利用CPG：sAAPF-pNA，酶变异体的吸附作用和对CPG结合肽的水解作用可通过一个简单的实验来测量。将一小体积的酶变异体贮存液加入含Tris缓冲剂和已抽气的CPG：sAAPF-pNA的烧瓶中。将烧瓶放入作曲形运动的摇床中摇90分钟，其中在不同的时间间隔停机几次(例如，在吸附水解的早期，如头20分钟，每2分钟停一次，而在实验的后期则每10分钟停一次)。CPG：sAAPF-pNA允许沉淀并在溶液中取样。实验方法及吸附作用和水解作用的计算均按以上Brode等人1992年描述的方法进行。
所有酶都检测了其抗自水解的稳定性而且在整个实验过程中不应有明显的自水解损失。因而酶的吸附作用可通过测溶液的消耗来确定。酶变异体初始浓度和在不同时刻所测得的酶浓度的差异体现了被吸附的酶变异体的量。从表面水解下来的pNA的量可通过测等份试样在410nm处的吸收得到。将试样量与烧瓶中剩余的量相加计算出水解下来的pNA的总量。这个值通过减去当没有酶时被pH8.6的Tris缓冲剂水解下来的pNA的量得到校正。依据酶的效率，这个本底水解作用占总水解作用的7-29％。
实施例6
可溶底物动力学分析
可溶底物sAAPF-pNA的水解速率通过在DU-70分光光度计上于410nm处测吸收升高与时间的函数得到检测。对每个动力学测量，酶浓度保持恒定，它被制成6-10纳摩尔，而底物浓度为90-700μMsAAPF-pNA。在900秒内每秒取一个吸收值且将数据转到LOTUS TM记录纸(Lotus Development Corporation，Cambridge，Massachusetts)上。动力学参数的分析可按标准的Lineweaver Burk分析进行，在LineweaverBurk分析中，曲线起始部分(通常第一分钟)的数据符合线性回归曲线，可得出vo。将vo与so数值以标准的倒数形式作图可得出KM和kcatI.蛋白酶K变异体实例
本发明中降低了对表面结合底物的吸附作用并提高了对表面结合底物的水解作用的蛋白酶K变异体在下面的表2-36中列出。描述特定突变时，首先给出的是野生型酶的原始氨基酸，其后为位置序号，最后是置换氨基酸。
表2
环区1-单突变变异体
Arg64Asp
Arg64Glu
Asp65Glu
Gly66Asn
Gly66Asp
Gly66Gln
Gly66Glu
Gly66Pro
Gly66Ser
Asn67Asp
Asn67Gln
Asn67Glu
Asn67Ser
Gly68Asn
Gly68Asp
Gly68Gln
Gly68Glu
Gly68Pro
Gly68Ser
Gly70Asn
Gly70Asp
Gly70Gln
Gly70Glu
Gly70Pro
Gly70Ser
Thr71Asn
Thr71Asp
Thr71Gln
Thr71Glu
Thr71Gly
Thr71Pro
Thr71Ser
表3环区1-双突变变异体Asn67Ser + Thr71AspGly66Ser + Thr71SerArg64Asp + Thr71AsnArg64Asp + Gly68SerGly66Glu + Gly70SerArg64Glu + Gly68SerArg64Glu + Gly66GlnAsn67Glu + Gly70AsnAsn67Gln + Thr71AspGly66Ser + Gly70AsnAsp65Glu + Gly70SerArg64Glu + Gly70AsnGly66Pro + Thr71AsnGly70Gln + Thr71GlnGly68Glu + Gly70AsnAsp65Glu + Thr71SerGly68Asp + Gly70GlnAsp65Glu + Gly68GlnGly70Gln + Thr71AspGly68Gln + Gly70ProAsn67Asp + Gly70GlnGly66Ser + Asn67GlnAsp65Glu + Gly68AsnGly68Asp + Gly70ProGly66Gln + Thr71ProAsp65Glu + Thr7lProArg64Asp + Gly68AsnGly68Asn + Thr71GluGly66Asn + Thr71GluGly68Ser + Thr71AspGly68Ser + Thr71GlyGly66Ser + Gly68GlnGly66Gln + Gly70SerGly66Glu + Gly70ProArg64Glu + Gly70ProAsp65Glu + Thr71GlyArg64Glu + Thr71GlyAsp65Glu + Gly70ProArg64Asp + Gly68GlnArg64Glu + Gly68ProGly66Asn + Gly70Ser
表4
环区1-三突变变异体Asn67Ser + Gly68Asp + Gly70SerAsn67Asp + Gly70Asn + Thr71GlyArg64Glu + Gly66Asn + Gly70ProGly66Asp + Gly68Ser + Gly70SerAsp65Glu + Gly70Gln + Thr71GlnGly66Glu + Gly68Pro + Gly70AsnArg64Asp + Gly66Ser + Gly70GlnGly66Gln + Asn67Gln + Gly68GluGly66Glu + Asn67Ser + Thr7lProGly66Asn + Gly68Pro + Thr71GlnArg64Glu + Gly66Gln + Gly68GlnGly66Asn + Gly68Ser + Gly70ProArg64Asp + Gly70Gln + Thr71GlyAsp65Glu + Gly68Gln + Gly70SerArg64Glu + Gly66Pro + Gly68SerGly66Gln + Asn67Gln + Thr71GlyArg64Glu + Gly68Pro + Thr71SerAsn67Asp + Gly70Ser + Thr71GlyArg64Glu + Gly70Pro + Thr71GlnAsp65Glu + Gly68Gln + Gly70GlnGly66Pro + Gly68Gln + Thr71ProGly66Glu + Asn67Gln + Gly70ProGly66Gln + Gly70Ser + Thr71GlnAsp65Glu + Gly68Gln + Gly70AsnArg64Glu + Gly66Ser + Thr71ProAsp65Glu + Gly68Ser + Gly70AsnAsp65Glu + Asn67Ser + Gly70SerArg64Glu + Gly66Ser + Gly70ProAsp65Glu + Gly70Gln + Thr71GlyAsp65Glu + Gly68Asn + Gly70AsnGly68Asn + Gly70Pro + Thr71AspArg64Asp + Gly68Ser + Gly70ProGly66Asn + Gly68Glu + Gly70GlnAsn67Glu + Gly68Gln + Gly70AsnGly66Glu + Gly68Pro + Gly70GlnAsn67Asp + Gly68Glu + Gly70SerAsn67Asp + Gly68Asp + Gly70AsnArg64Asp + Asp65Glu + Asn67SerArg64Asp + Asp65Glu + Gly70ProArg64Glu + Asp65Glu + Asn67Gln
表5
环区1-四突变变异体Asp65Glu + Gly68Pro + Gly70Asn + Thr71AsnAsp65Glu + Asn67Gln + Gly68Gln + Gly70SerGly66Glu + Asn67Gln + Gly68Asn + Gly70GlnAsp65Glu + Gly66Asn + Gly70Ser + Thr71GlnAsn67Gln + Gly68Glu + Gly70Gln + Thr71GlyArg64Glu + Gly66Gln + Gly70Asn + Thr71GlnGly66Pro + Gly68Ser + Gly70Asn + Thr71GlnArg64Glu + Gly68Pro + Gly70Gln + Thr71ProGly66Glu + Gly68Pro + Gly70Asn + Thr71ProAsp65Glu + Gly66Glu + Asn67Ser + Gly70ProAsp65Glu + Gly66Asp + Gly68Asn + Thr71SerAsp65Glu + Gly66Glu + Gly68Pro + Gly70SerAsp65Glu + Gly66Glu + Gly70Asn + Thr71GlnArg64Asp + Asp65Glu + Asn67Gln + Gly70AsnArg64Asp + Asp65Glu + Gly70Pro + Thr71GlnGly66Glu + Asn67Asp + Gly68Ser + Gly70SerGly66Glu + Asn67Glu + Gly70Asn + Thr71SerAsp65Glu + Gly66Asp + Asn67Glu + Gly68SerArg64Glu + Asp65Glu + Gly66Asp + Gly68ProGly66Glu + Asn67Asp + Gly68Asp + Gly70AsnAsp65Glu + Asn67Glu + Gly68Ser + Gly70GlnAsp65Glu + Asn67Asp + Gly68Gln + Gly70SerAsp65Glu + Asn67Glu + Gly68Gln + Gly70AsnAsp65Glu + Gly66Asn + Asn67Glu + Gly70ProAsp65Glu + Gly66Asp + Gly68Asp + Thr71AsnAsp65Glu + Gly66Glu + Asn67Gln + Gly68Asp
表6环区2-单突变变异体Val 95AlaVal 95AsnVal 95AspVal 95CysVal 95GlnVal 95GluVal 95GlyVal 95HisVal 95MetVal 95ProVal 95SerVal 95ThrLeu 96AlaLeu 96AsnLeu 96AspLeu 96CysLeu 96GlnLeu 96GluLeu 96GlyLeu 96HisLeu 96IleLeu 96MetLeu 96ProLeu 96SerLeu 96ThrLeu 96ValAsp 97GluAsp 98GluAsn 99AspAsn 99GlnAsn 99GluAsn 99SerGly100AsnGly100AspGlyl00GlnGly100GluGly100ProGly100SerSer101AspSer101GluGly102AsnGly102AspGly102GlnGly102GluGly102ProGly102SerGln103AsnGln103AspGln103GluGln103SerTyr104AlaTyr104AsnTyr104AspTyr104CysTyr104GlnTyr104GluTyr104GlyTyr104HisTyr104IleTyr104LeuTyr104MetTyr104ProTyr104SerTyr104ThrTyr104ValSer105AspSer105GluThr106AsnThr106AspThr106GlnThr106GluThr106GlyThr106ProThr106SerIle107AlaIle107AsnIle107AspIle107CysIle107GlnIle107GluIle107GlyIle107HisIle107LeuIle107MetIle107ProIle107Ser
20Ile107ThrIle107Val
表7环区2-双突变变异体Val 95Gln + Ser101GluTyr104His + Ile107AspVal 95Glu + Ile107AsnAsn 99Gln + Gln103AspLeu 96Gly + Ser105GluVal 95Met + Gln103AspSer101Asp + Ile107ThrVal 95His + Asp 97GluAsp 98Glu + Tyr104LeuLeu 96Cys + Asp 97GluGln103Glu + Tyr104GlnGly102Ser + Tyr104GlyLeu 96Ala + Thr106ProThr106Gln + Ile107AsnAsn 99Gln + Ile107AspAsn 99Glu + Thr106GlyGly102Pro + Gln103AspAsn 99Asp + Tyr104ThrLeu 96Ile + Ser101GluVal 95Gly + Gln103SerTyr104Leu + Ser105GluGly102Asn + Tyr104ProLeu 96Asp + Ile107LeuAsp 98Glu + Gly102AsnLeu 96Pro + Gly100GluSer101Asp + Thr106SerGly100Ser + Gln103AsnGly102Pro + Tyr104ThrLeu 96Glu + Ile107LeuLeu 96Ile + Gty100AspGly100Pro + Ile107SerAsp 97Glu + Asn 99GlnAsp 97Glu + Ile107CysGly102Ser + Gln103GluGly100Asn + Tyr104AspGly100Pro + Thr106AspVal 95Pro + Asp 97GluVal 95Thr + Gly100GluThr106Pro + Ile107GluSer101Asp + Gly102AsnSer105Glu + Ile107ThrAsn 99Gln + Ser105AspGln103Ser + Ile107MetLeu 96Asp + Thr106GlyVal 95Thr + Gly102AspVal 95Ala + Ser105Asp
21Gly100Asn + Gln103SerGly102Asp + Thr106GlnLeu 96Asn + Ser105AspGly100Glu + Gly102ProGln103Asp + Ile107HisTyr104Ala + Ile107ProAsp 97Glu + Ile107ProAsn 99Glu + Tyr104AsnVal 95Thr + Asp 98GluGly100Gln + Thr106GlyAsn 99Glu + Gln103SerVal 95His + Ser105AspGly102Pro + Ile107AlaAsp 97Glu + Gly100Gln
表8
环区2-三突变变异体Val 95Gln + Leu 96Thr + Ser101GluSer101Asp + Thr106Ser + Ile107LeuVal 95Asn + Leu 96Asn + Asp 98GluGln103Glu + Tyr104Ser + Ile107SerVal 95Cys + Leu 96Glu + Gly102SerVal 95His + Tyr104Thr + Ser105GluLeu 96Pro + Asn 99Asp + Gln103SerVal 95Ser + Leu 96Asp + Gln103SerLeu 96Cys + Gly102Gln + Ile107LeuVal 95Ser + Leu 96His + Thr106ProLeu 96Gln + Gly102Pro + Ser105AspLeu 96Met + Asn 99Ser + Ser105AspLeu 96His + Ser101Asp + Tyr104ValAsn 99Asp + Gly100Asn + Gly102SerVal 95Gln + Gly100Asp + Thr106ProLeu 96Asn + Tyr104Glu + Ile107GlnAsn 99Asp + Gly102Ser + Tyr104HisVal 95Met + Leu 96Gly + Gly100ProVal 95Ala + Asp 98Glu + Asn 99SerAsp 97Glu + Tyr104Thr + Ile107HisLeu 96Thr + Gly102Gln + Gln103AspTyr104Met + Thr106Glu + Ile107LeuGly100Ser + Gln103Asn + Tyr104IleGln103Asn + Tyr104Pro + Thr106GluVal 95Met + Asp 98Glu + Asn 99GlnLeu 96His + Asp 97Glu + Ile107MetGly100Asn + Gln103Ser + Ser105AspGly102Asn + Gln103Glu + Thr106ProVal 95Met + Gly100Ser + Gly102AspVal 95Asp + Leu 96Met + Tyr104AsnGln103Glu + Tyr104Cys + Ile107AsnVal 95Met + Leu 96Ile + Tyr104MetVal 95Ser + Ser101Glu + Thr106GlyVal 95Pro + Asn 99Asp + Gly100SerGly100Ser + Gln103Glu + Ile107ValGln103Asp + Tyr104Val + Ile107HisGly100Gln + Tyr104Met + Thr106AspAsn 99Asp + Gly100Ser + Tyr104HisSer101Asp + Gly102Asp + Gln103SerAsp 98Glu + Asn 99Asp + Tyr104AlaAsp 98Glu + Asn 99Glu + Gly100GlnGly102Asp + Gln103Asp + Thr106GlnTyr104Glu + Ser105Asp + Thr106ProAsp 97Glu + Asp 98Glu + Tyr104ThrAsp 97Glu + Asp 98Glu + Asn 99AspAsn 99Asp + Gly100Asp + Ser101GluGln103Glu + Tyr104Asp + Ile107GluLeu 96Glu + Asp 97Glu + Gly102AspLeu 96Glu + Ser101Asp + Gln103AsnAsp 97Glu + Gly100Asp + Ile107HisAsp 97Glu + Asn 99Ser + Gly100AspGln103Asp + Ser105Glu + Thr106AsnLeu 96Ser + Ser105Asp + Ile107AspAsp 97Glu + Ser101Glu + Gln103SerAsp 97Glu + Ser101Glu + Tyr104MetAsn 99Asp + Gly100Ser + Ser101AspGln103Asp + Tyr104His + Ile107GluAsp 97Glu + Gly102Glu + Tyr104ProVal 95Asp + Gly102Glu + Tyr104GlyVal 95Asp + Asp 97Glu + Ile107Asn
表9
环区2-四突变变异体Val 95Gln + Asp 97Glu + Gly100Ser + Ile107GlnVal 95Ser + Leu 96Pro + Asp 97Glu + Gln103AsnLeu 96Gln + Gly100Ser + Ser101Asp + Thr106AsnVal 95Cys + Asp 97Glu + Gly100Gln + Gly102SerLeu 96Pro + Gly102Asn + Ser105Asp + Ile107GlnVal 95Ser + Gly100Gln + Ser101Glu + Ile107AsnLeu 96Met + Ser101Glu + Gln103Asn + Thr106ProAsn 99Asp + Gly100Pro + Gly102Ser + Tyr104AlaGly100Pro + Gly102Pro + Thr106Gly + Ile107GluVal 95Ser + Gly100Gln + Ser101Asp + Gly102ProVal 95Gly + Leu 96Val + Thr106Pro + Ile107AspVal 95Gln + Leu 96Val + Asn 99Asp + Ile107ThrLeu 96Ser + Asp 97Glu + Thr106Gln + Ile107MetLeu 96Thr + Asn 99Gln + Tyr104Val + Ile107CysAsn 99Glu + Gly100Gln + Gly102Asn + Gln103SerVal 95Gly + Ser101Glu + Gln103Ser + Thr106GlyVal 95Asn + Leu 96Gln + Asp 98Glu + Gly100AsnVal 95Cys + Gly102Pro + Tyr104Val + Ile107AspVal 95Met + Leu 96Met + Gly102Glu + Tyr104ThrVal 95Asp + Leu 96Cys + Thr106Ser + Ile107GlyLeu 96Met + Gly100Glu + Ser101Glu + Tyr104CysAsp 98Glu + Asn 99Glu + Gly102Asn + Ile107SerVal 95Ser + Asp 97Glu + Asp 98Glu + Asn 99SerVal 95Gly + Asp 97Glu + Asp 98Glu + Tyr104IleLeu 96Gly + Asp 97Glu + Asp 98Glu + Asn 99AspAsp 98Glu + Asn 99Glu + Gly100Glu + Thr106GlyLeu 96Glu + Asp 97Glu + Ser101Asp + Tyr104IleAsp 97Glu + Gly100Asp + Ser101Glu + Tyr104LeuAsp 97Glu + Asp 98Glu + Gly100Glu + Tyr104MetGly100Glu + Ser101Asp + Gly102Asp + Ile107GlyVal 95Met + Leu 96Glu + Asp 97Glu + Gly102AspAsp 97Glu + Gly100Glu + Tyr104Ala + Thr106GlnAsp 97Glu + Asn 99Glu + Tyr104His + Thr106GlyGly102Ser + Gln103Asp + Ser105Asp + Thr106SerAsp 97Glu + Ser101Asp + Gly102Pro + Tyr104IleAsn 99Asp + Gly100Gln + Ser101Asp + Thr106ProAsn 99Asp + Gly100Ser + Ser101Asp + Ile107CysGly102Ser + Gln103Glu + Ser105Asp + Ile107AspAsp 98Glu + Gly100Glu + Ser101Glu + Gly102GlnVal 95Gly + Asp 98Glu + Asn 99Glu + Ser101AspAsp 97Glu + Gly102Asp + Gln103Ser + Ile107MetLeu 96Ser + Asp 97Glu + Gly100Glu + Gly102GluVal 95Thr + Leu 96Glu + Asp 98Glu + Ser101AspGly102Glu + Tyr104Ala + Ser105Asp + Thr106AspLeu 96Met + Ser101Asp + Gln103Glu + Ile107GlnSer101Asp + Gln103Asp + Tyr104Ser + Ile107ThrAsp 98Glu + Gly100Asn + Ser101Glu + Tyr104ValVal 95Glu + Gly102Glu + Gln103Asn + Tyr104GluVal 95Thr + Ser101Glu + Gln103Asp + Ile107GluSer101Glu + Gln103Asp + Tyr104Met + Ile107GluVal 95Glu + Ser101Asp + Tyr104His + Ile107GluVal 95Gly + Asp 97Glu + Gly102Glu + Thr106GluVal 95Ser + Leu 96Asp + Asn 99Gln + Gln103GluLeu 96Glu + Gly102Asn + Tyr104Gln + Thr106GluVal 95Asp + Asp 98Glu + Gly100Asp + Thr106GlyLeu 96Glu + Gly102Asp + Gln103Ser + Ser105GluGly100Ser + Ser101Glu + Gln103Asp + Ser105AspVal 95Met + Gly100Glu + Gly102Asp + Ile107AspLeu 96Ala + Asp 97Glu + Ser101Asp + Ile107AspLeu 96Gln + Asp 97Glu + Gln103Asp + Ile107Glu
表10环区3-单突变变异体Leu133AlaLeu133AsnLeu133AspLeu133CysLeu133GlnLeu133GluLeu133GlyLeu133HisLeu133IleLeu133MetLeu133ProLeu133SerLeu133ThrLeu133ValGly134AsnGly134AspGly134GlnGly134GluGly134ProGly134SerGly135AsnGly135AspGly135GlnGly135GluGly135ProGly135SerGly136AsnGly136AspGly136GlnGly136GluGly136ProGly136SerTyr137AlaTyr137AsnTyr137AspTyr137CysTyr137GlnTyr137GluTyr137GlyTyr137HisTyr137IleTyr137LeuTyr137MetTyr137ProTyr137SerTyr137ThrTyr137ValSer138AspSer138GluSer139AspSer139GluSer140AspSer140Glu
表11环区3-双突变变异体Gly134Asn + Ser140AspLeu133Ala + Gly135GluLeu133Thr + Ser139AspLeu133Gln + Ser140AspGly136Gln + Ser138GluGly134Ser + Ser138AspGly136Pro + Ser139AspGly135Asn + Ser138AspGly135Glu + Gly136ProLeu133Ile + Ser140AspLeu133Cys + Ser140AspGly134Asn + Gly135ProLeu133Cys + Gly136AspTyr137Asn + Ser139AspGly136Asn + Ser140GluGly134Glu + Gly135ProTyr137Met + Ser140AspGly135Gln + Ser139GluTyr137Thr + Ser138GluLeu133Asn + Ser139GluGly135Ser + Gly136GlnLeu133Gly + Gly136SerLeu133Ala + Tyr137HisLeu133Val + Gly136GluTyr137Ile + Ser139AspLeu133Ile + Tyr137GlnGly136Ser + Ser140AspGly134Asn + Ser138GluGly134Ser + Ser138GluGly135Ser + Ser138AspGly136Ser + Ser140GluGly136Ser + Ser138GluLeu133Glu + Gly134AsnLeu133Glu + Gly135GlnGly135Asn + Tyr137GluTyr137Thr + Ser139GluTyr137Ala + Ser139GluGly134Gln + Tyr137GlyGly135Pro + Tyr137HisLeu133Pro + Ser138GluLeu133Thr + Gly135SerGly136Pro + Ser138GluGly134Ser + Ser140AspLeu133Met + Tyr137AlaTyr137Val + Ser138GluGly134Pro + Ser140GluLeu133Thr + Tyr137SerGly135Glu + Gly136AsnLeu133Ala + Ser140GluGly134Gln + Ser139GluLeu133Pro + Tyr137GlyLeu133Val + Gly136SerGly136Asp + Tyr137ProGly136Asn + Tyr137ValLeu133Ala + Ser138AspGly135Gln + Gly136ProTyr137Met + Ser138GluLeu133Gln + Tyr137AspTyr137Ser+Ser138GluG1y134Asp+Gly135Pro
表12环区3-三突变变异体Leu133Cys + Gly135Gln + Tyr137GluLeu133Ile + Gly134Ser + Gly136GluLeu133Gly + Gly135Glu + Tyr137ValLeu133Gln + Gly135Gln + Ser139AspLeu133Asp + Gly135Pro + Tyr137AsnLeu133Val + Gly135Gln + Gly136SerGly135Asn + Tyr137Ile + Ser139GluLeu133Met + Gly136Pro + Ser140AspGly134Pro + Gly136Pro + Ser138GluGly135Asn + Tyr137Met + Ser138AspLeu133Ser + Gly136Asn + Tyr137ValGly134Glu + Gly135Ser + Gly136ProGly136Asn + Tyr137Met + Ser140GluLeu133Ile + Gly136Pro + Tyr137GluLeu133Met + Gly136Gln + Tyr137GlnLeu133Val + Gly135Pro + Ser139AspLeu133Asp + Gly135Gln + Tyr137CysGly135Pro + Tyr137Met + Ser138GluGly136Gln + Tyr137Gly + Ser140AspLeu133Ser + Gly134Pro + Ser140AspLeu133Thr + Gly134Asp + Gly135ProLeu133Ile + Gly135Asn + Ser139GluGly135Glu + Gly136Pro + Tyr137HisGly135Asn + Gly136Pro + Ser140GluLeu133Met + Gly134Ser + Gly136GlnGly134Pro + Gly135Asn + Ser140GluGly135Gln + Gly136Gln + Ser138AspGly136Ser + Tyr137Met + Ser138GluGly134Pro + Gly136Ser + Ser139AspGly134Ser + Tyr137Thr + Ser138AspLeu133Thr + Gly134Pro + Ser140AspGly134Gln + Gly135Pro + Ser140GluGly134Pro + Tyr137His + Ser138AspLeu133Met + Gly134Gln + Ser139AspLeu133Ser + Gly134Asn + Ser138GluGly136Gln + Tyr137Ala + Ser139GluLeu133Ile + Gly136Asn + Ser139AspLeu133Ala + Gly135Asp + Gly136ProLeu133Asn + Ser138Asp + Ser139GluGly136Asn + Ser138Asp + Ser139AspGly135Asn + Ser138Asp + Ser139GluLeu133Asp + Gly134Asp + Tyr137HisLeu133Asp + Gly134Glu + Gly135ProLeu133Gly + Gly136Glu + Tyr137AspTyr137Ser + Ser139Glu + Ser140AspGly134Gln + Ser139Asp + Ser140AspGly136Asn + Ser139Asp + Ser140GluTyr137Pro + Ser139Asp + Ser140AspGly135Ser + Tyr137Glu + Ser138GluSer138Asp + Ser139Asp + Ser140AspTyr137Glu + Ser138Asp + Ser139GluTyr137Ala + Ser138Asp + Ser140AspTyr137Thr + Ser138Asp + Ser140GluTyr137Gln + Ser138Asp + Ser140AspLeu133Thr + Ser138Asp + Ser140GluGly135Ser + Ser138Asp + Ser140GluGly136Gln + Ser138Asp + Ser140AspGly135Ser + Ser138Glu + Ser140AspGly136Gln + Ser138Glu + Ser140AspLeu133Val + Ser138Glu + Ser140Glu
表13
环区3-四突变变异体Leu133Ala + Gly134Asn + Tyr137Ser + Ser138AspGly134Glu + Gly135Asn + Gly136Asn + Tyr137CysLeu133Cys + Gly135Asn + Gly136Gln + Ser138AspLeu133Pro + Gly134Asn + Gly135Ser + Ser139AspLeu133Val + Gly134Gln + Gly136Gln + Ser140GluLeu133Asp + Gly134Gln + Gly136Gln + Tyr137ThrLeu133His + Gly135Gln + Gly136Pro + Tyr137SerLeu133Gly + Gly134Ser + Tyr137Ala + Ser138AspLeu133Gln + Gly134Gln + Gly135Asn + Ser140GluLeu133Ser + Gly135Ser + Tyr137Pro + Ser139GluLeu133Asn + Gly134Asn + Gly136Glu + Tyr137CysLeu133Met + Gly134Asn + Gly135Gln + Ser138GluLeu133Asn + Gly134Ser + Tyr137Val + Ser138GluGly135Asn + Gly136Pro + Tyr137Cys + Ser139AspLeu133Ile + Gly135Asp + Gly136Asn + Tyr137IleGly134Asp + Gly135Glu + Gly136Gln + Tyr137ThrLeu133Gln + Gly134Glu + Gly135Asp + Tyr137ThrGly134Ser + Tyr137Val + Ser138Glu + Ser139GluGly134Ser + Tyr137Val + Ser138Asp + Ser139GluLeu133His + Gly135Gln + Ser138Asp + Ser139AspLeu133Gly + Gly135Pro + Ser138Glu + Ser139AspLeu133Val + Gly135Pro + Ser138Asp + Ser139GluLeu133Ser + Gly134Gln + Gly135Asp + Gly136GluLeu133Ile + Gly134Gln + Ser139Asp + Ser140AspLeu133Cys + Tyr137Asn + Ser139Asp + Ser140GluLeu133Ser + Tyr137His + Ser139Glu + Ser140GluGly134Pro + Gly136Gln + Ser139Asp + Ser140GluGly134Gln + Gly135Ser + Ser139Asp + Ser140AspLeu133Cys + Gly134Pro + Ser139Glu + Ser140AspLeu133Ala + Gly136Asn + Tyr137Glu + Ser138AspGly136Pro + Ser138Asp + Ser139Glu + Ser140AspTyr137Leu + Ser138Asp + Ser139Glu + 5er140GluGly135Asn + Ser138Asp + Ser139Asp + Ser140GluLeu133Gly + Ser138Asp + Ser139Glu + Ser140GluLeu133Ser + Tyr137Asp + Ser138Asp + Ser139GluGly136Gln + Tyr137Asp + Ser138Asp + Ser139AspGly136Asn + Tyr137Asp + Ser138Asp + Ser139GluGly134Glu + Gly135Asp + Gly136Asp + Tyr137IleLeu133Gln + Tyr137Val + Ser138Glu + Ser140AspGly134Pro + Tyr137Gly + Ser138Asp + Ser140AspLeu133His + Tyr137Leu + Ser138Glu + Ser140GluGly134Gln + Gly135Ser + Ser138Glu + Ser140GluLeu133Cys + Gly136Ser + Ser138Glu + Ser140GluGly135Gln + Gly136Gln + Ser138Asp + Ser140AspGly134Pro + Tyr137His + Ser138Asp + Ser140GluLeu133Gln + Gly134Pro + Gly136Glu + Ser138AspLeu133His + Gly134Pro + Gly136Asp + Ser138GluLeu133Ile + Gly135Glu + Tyr137Glu + Ser138GluGly134Asn + Gly135Asp + Tyr137Asp + Ser138GluGly135Gln + Tyr137Glu + Ser138Asp + Ser140GluGly136Asn + Tyr137Glu + Ser138Asp + Ser140GluLeu133Asn + Tyr137Asp + Ser138Glu + Ser140AspLeu133Cys + Gly136Pro + Tyr137Glu + Ser139AspGly134Glu + Gly135Pro + Gly136Asp + Tyr137GluGly136Pro + Tyr137Glu + Ser139Asp + Ser140AspGly135Ser + Tyr137Glu + Ser139Glu + Ser140GluLeu133Pro + Gly134Glu + Gly136Asp + Tyr137CysGly134Gln + Gly136Asp + Ser138Glu + Ser139AspGly135Ser + Gly136Glu + Ser138Glu + Ser139AspGly134Asp + Gly135Glu + Gly136Ser + Ser138Asp
表14环区4-单突变变异体Gly160AsnGly160AspGly160GlnGly160GluGly160ProGly160SerAsn161AspAsn161GlnAsn161GluAsn161SerAsn162AspAsn162GlnAsn162GluAsn162SerAsn163AspAsn163GlnAsn163GluAsn163SerAla164AsnAla164AspAla164GlnAla164GluAla164GlyAla164HisAla164ProAla164SerAla164ThrAsp165GluAla166AsnAla166AspAla166GlnAla166GluAla166GlyAla166HisAla166ProAla166SerAla166ThrArg167AspArg167GluAsn168AspAsn168GlnAsn168GluAsn168SerTyr169AlaTyr169AsnTyr169AspTyr169CysTyr169GlnTyr169GluTyr169GlyTyr169HisTyr169IleTyr169LeuTyr169MetTyr169ProTyr169SerTyr169ThrTyr169ValSer170AspSer170Glu
表15环区4-双突变变异体Gly160Gln + Asp165GluAsn161Ser + Asn162SerAla164Thr + Asn168AspGly160Asn + Ser170GluAsn161Gln + Asn163GluAsn162Ser + Ser170AspGly160Gln + Asn168GluAla166Gly + Ser170AspAsn161Glu + Asn162SerAsn162Gln + Ser170AspAla166Thr + Ser170GluAsn162Ser + Ala164ProGly160Asn + Asn161GlnGly160Glu + Ala166SerAla166Gly + Ser170GluAla164His + Ala166GlyAla164Asn + Asp165GluAsn162Asp + Asn168SerAla164Pro + Asn168AspAsn162Asp + Ala164AsnTyr169Thr + Ser170AspAla164Glu + Tyr169HisAsp165Glu + Tyr169HisAsn162Glu + Asn168SerGly160Asp + Tyr169MetAsn162Glu + Ala166AsnAsn161Glu + Ala166ThrAsp165Glu + Ala166AsnAsn161Gln + Tyr169ProAsn162Asp + Ala164ThrAsn162Ser + Arg167GluAla166Pro + Arg167AspAla164Thr + Ser170AspAsn163Asp + Ala164AsnAsp165Glu + Asn168GlnAsn163Asp + Tyr169ThrAsn168Asp + Tyr169GlyArg167Asp + Asn168SerAsn161Ser + Arg167GluAsn161Ser + Ser170GluGly160Asp + Ala166GlyAla164Asn + Ala166GlnGly160Asn + Asn161GluAsn162Glu + Ala166SerAla166His + Asn168AspGly160Pro + Ala164GlyGly160Asn + Ala164GlyAla164Gln + Ser170AspAsn161Glu + Ala164ThrGly160Glu + Ala166GlyAla166Pro + Ser170AspAla166Pro + Tyr169AlaGly160Ser + Arg167GluGly160Asp + Ala164SerAsn161Gln + Ala164ProAsn163Glu + Ala166GlnAla164Ser + Asn168GlnGly160Glu + Ala164SerGly160Asp + Ala166GlnAla164Thr + Asp165Glu
表16
环区4-三突变变异体Gly160Gln + Asn161Ser + Asp165GluAla166Gly + Arg167Glu + Tyr169CysAsn162Ser + Ala166Asn + Ser170AspGly160Gln + Asn162Asp + Asn168SerAla164Asn + Asn168Gln + Ser170GluAla164Asp + Ala166Thr + Asn168SerAsn163Gln + Ala166Gly + Arg167GluAsn162Ser + Ala166His + Asn168GlnAsp165Glu + Ala166Thr + Asn168GlnAsn163Ser + Ala164Pro + Asn168AspGly160Glu + Ala164His + Tyr169GlnAsn161Gln + Asn163Ser + Ser170GluAla166Thr + Tyr169Leu + Ser170AspGly160Ser + Ala164Asn + Asn168GluAsn161Gln + Asn162Glu + Asn163SerAsn163Ser + Ala166Ser + Arg167GluAla164Thr + Ala166Gly + Arg167GluAla164Ser + Ala166Gly + Ser170GluAsn162Gln + Ala164Gln + Ala166ProAsn162Glu + Asn163Gln + Ala164GlyAsn161Asp + Ala164Asn + Tyr169ValGly160Asn + Ala164Glu + Ala166ProAsn162Gln + Ala164Pro + Arg167AspAsn163Ser + Ala164Ser + Tyr169GlyAla166Asn + Arg167Glu + Asn168SerAsn163Gln + Asp165Glu + Ala166SerAsn162Ser + Ala164His + Asn168AspAla164Ser + Arg167Asp + Asn168AspAsn162Ser + Arg167Asp + Asn168AspAsn161Asp + Asn162Glu + Asn163GlnAsn161Asp + Asn162Glu + Asn168GlnGly160Glu + Asn161Glu + Ala166AsnGly160Glu + Asn161Glu + Ala164SerGly160Pro + Asn162Asp + Asn163AspAsn162Asp + Asn163Glu + Ala166AsnAsn161Glu + Asn162Asp + Asn163GluAsn162Glu + Asn163Ser + Ala164AspAsp165Glu + Arg167Asp + Tyr169ProAsp165Glu + Arg167Asp + Asn168AspAla164Gln + Arg167Asp + Tyr169AspAsn161Asp + Asn162Asp + Ala164AspAsn161Asp + Asn163Asp + Tyr169ThrAsn161Asp + Asn163Asp + Ala164AsnGly160Glu + Asn162Glu + Asn163GlnAla164Asp + Asp165Glu + Arg167GluAla164Pro + Asp165Glu + Asn168AspGly160Asp + Asn162Glu + Ala164GluGly160Pro + Asn168Glu + Ser170GluAla164Glu + Arg167Glu + Asn168GluAsn161Glu + Asn163Glu + Asp165GluGly160Asp + Asn162Glu + Asp165GluAsp165Glu + Ala166Gly + Tyr169GluGly160Glu + Asn161Asp + Asp165GluArg167Asp + Asn168Gln + Ser170AspAsn162Gln + Arg167Asp + Ser170GluAsn163Gln + Arg167Glu + Ser170GluAsn162Asp + Asp165Glu + Asn168GluAsn162Asp + Asp165Glu + Arg167GluGly160Asp + Asn168Gln + Ser170GluGly160Asp + Asp165Glu + Tyr169Gly
表17
环区4-四突变变异体Gly160Gln + Asn161Ser + Asn162Ser + Asp165GluGly160Glu + Asn161Ser + Asn162Gln + Asn168SerAsn161Ser + Asn163Asp + Ala164Asn + Tyr169ThrGly160Ser + Asp165Glu + Ala166Asn + Tyr169LeuGly160Gln + Ala166Ser + Arg167Asp + Tyr169HisGly160Ser + Asn163Ser + Ala166Gly + Ser170AspAsn161Gln + Asn163Gln + Ala164Glu + Ala166GlyAsn161Glu + Asn163Gln + Ala166His + Tyr169AsnAsn163Gln + Ala166His + Arg167Asp + Asn168GlnGly160Gln + Asn162Ser + Asn163Asp + Tyr169GlnAsn162Glu + Ala166Gln + Asn168Ser + Tyr169GlyAsn163Glu + Ala166His + Asn168Gln + Tyr169GlnGly160Asn + Asn161Ser + Ala164Thr + Ala166SerAsn161Gln + Asn162Ser + Asn168Glu + Tyr169SerAsn161Gln + Ala166His + Arg167Glu + Tyr169AlaAsn161Gln + Ala164Gly + Ala166Gln + Tyr169HisAla164Gln + Asp165Glu + Asn168Ser + Tyr169MetAla164Gln + Ala166His + Arg167Glu + Asn168GluGly160Asn + Asn162Ser + Ala164Glu + Asp165GluAsnl61Asp + Asn162Asp + Ala164Asn + Ala166SerAsn161Asp + Asn162Asp + Ala164Asn + Ala166ThrAla164Gln + Ala166Asn + Asn168Asp + Tyr169AspGly160Pro + Asn163Asp + Ala164Asp + Ala166ThrAsn161Ser + Asn163Asp + Ala164Asp + Asn168GlnAsn162Asp + Asn163Asp + Ala166Pro + Tyr169MetAsn161Ser + Asn163Gln + Tyr169Asp + Ser170AspAsn161Ser + Ala166His + Tyr169Asp + Ser170GluAsn161Glu + Asn162Asp + Asn163Glu + Tyr169CysAsn161Glu + Asn162Glu + Asn163Asp + Tyr169AlaAsp165Glu + Ala166Gly + Arg167Asp + Asn168GlnAsp165Glu + Arg167Asp + Asn168Gln + Tyr169ValAsn161Asp + Asn163Glu + Ala164Asn + Ala166GlyAsn163Gln + Ala164Asp + Asp165Glu + Arg167AspAsp165Glu + Ala166Asn + Asn168Asp + Tyr169GluAsn163Gln + Asp165Glu + Arg167Glu + Tyr169AspAsp165Glu + Arg167Asp + Asn168Ser + Tyr169AspAsn163Ser + Asp165Glu + Arg167Asp + Tyr169GluAsn163Gln + Ala166Thr + Asn168Glu + Ser170GluAsn161Gln + Ala166Gly + Asn168Asp + Ser170GluAsn163Glu + Asp165Glu + Ala166Gly + Tyr169LeuGly160Pro + Asn162Gln + Asn163Asp + Asp165GluGly160Glu + Asn163Gln + Asn168Asp + Tyr169GluGly160Asp + Ala164Glu + Asp165Glu + Tyr169SerAsn161Glu + Ala164Asp + Asp165Glu + Tyr169GlnGly160Asp + Asn162Asp + Ala166Gly + Tyr169AspAsn161Glu + Asn162Glu + Asp165Glu + Ala166AsnAsn161Asp + Asn162Asp + Asp165Glu + Ala166GlnAsn161Asp + Asn163Asp + Asp165Glu + Asn168GlnGly160Asp + Asn161Ser + Asn163Glu + Tyr169GlyGly160Asp + Asn162Asp + Asp165Glu + Asn168GlnAsn161Ser + Arg167Glu + Tyr169Thr + Ser170AspAsn161Gln + Arg167Glu + Asn168Gln + Ser170AspGly160Asp + Asn163Asp + Asp165Glu + Tyr169AlaGly160Ser + Asn162Glu + Ala164Pro + Asp165GluGly160Glu + Arg167Glu + Asn168Glu + Tyr169GlnAsn161Ser + Ala164Asp + Arg167Glu + Tyr169AsnAsn162Ser + Asn163Asp + Ala164Asp + Asn168AspAsn163Glu + Asp165Glu + Ala166Asn + Arg167GluAsn163Asp + Asp165Glu + Arg167Asp + Asn168SerGly160Glu + Ala164Gly + Asn168Asp + Ser170Asp
表18环区5-单突变变异体Ser190AspSer190GluSer191AspSer191GluPhe192AlaPhe192AsnPhe192AspPhe192CysPhe192GlnPhe192GluPhe192GlyPhe192HisPhe192IlePhe192LeuPhe192MetPhe192ProPhe192SerPhe192ThrPhe192TyrPhe192ValSer193AspSer193GluAsn194AspAsn194GlnAsn194GluAsn194Ser
表19环区5-双突变变异体Ser190Asp + Phe192LeuSer190Glu + Phe192GlnPhe192Glu + Asn194GlnPhe192Tyr + Asn194GluSer190Asp + Phe192IleSer191Glu + Phe192SerSer191Asp + Phe192GlySer191Asp + Asn194SerSer191Glu + Pne192ThrSer191Glu + Asn194GlnSer191Glu + Phe192HisSer191Asp + Phe192AsnSer190Asp + Phe192GlySer191Asp + Asn194GlnSer191Asp + Phe192AlaSer190Glu + Phe192CysSer190Glu + Phe192IleSer191Asp + Phe192SerSer190Asp + Asn194GlnSer191Glu + Phe192CysPhe192Ser + Asn194GluSer191Glu + Phe192GlyPhe192Gly + Asn194GlnPhe192Cys + Asn194GlnPhe192Asn + Asn194AspSer190Glu + Phe192TyrSer191Glu + Phe192AlaSer190Glu + Asn194SerPhe192Ile + Asn194SerSer191Glu + Phe192MetSer190Asp + Phe192MetSer190Asp + Asn194SerPhe192Gly + Asn194AspPhe192His + Asn194SerSer190Glu + Asn194GlnPhe192Ala + Asn194GluPhe192Thr + Asn194GluSer190Glu + Phe192SerSer191Glu + Phe192IlePhe192Val + Asn194GluPhe192Thr + Asn194AspSer191Glu + Phe192ProSer191Asp + Phe192LeuPhe192Ile + Asn194GluSer190Asp + Phe192ThrSer191Asp + Phe192IlePhe192Thr + Asn194GlnPhe192Ala + Asn194GlnSer191Asp + Phe192TyrSer190Glu + Phe192ThrPhe192Leu + Asn194GluPhe192His + Asn194GlnSer190Asp + Phe192TyrPhe192Val + Asn194SerPhe192Asp + Asn194GlnPhe192Gly + Asn194SerSer191Asp + Phe192GlnPhe192Asp + Asn194SerPhe192Thr + Asn194SerSer190Glu + Phe192Ala
表20
环区5-三突变变异体Ser191Asp + Phe192Met + Asn194GlnSer191Glu + Phe192Ala + Asn194GlnSer191Asp + Phe192Leu + Asn194SerSer191Glu + Phe192Asn + Asn194GlnSer190Asp + Phe192Gln + Asn194GlnSer191Asp + Phe192Ile + Asn194GlnSer190Asp + Phe192Asn + Asn194SerSer191Glu + Phe192Ile + Asn194SerSer190Asp + Phe192Leu + Asn194GlnSer190Asp + Phe192Val + Asn194SerSer190Glu + Phe192Gln + Asn194SerSer191Asp + Phe192Val + Asn194SerSer191Glu + Phe192Ala + Asn194SerSer191Glu + Phe192Cys + Asn194GlnSer190Glu + Phe192Val + Asn194SerSer190Glu + Phe192Ile + Asn194GlnSer190Glu + Phe192Ser + Asn194SerSer190Glu + Phe192Tyr + Asn194GlnSer191Asp + Phe192Ile + Asn194SerSer190Asp + Ser191Glu + Asn194SerSer190Asp + Ser191Asp + Phe192MetSer190Glu + Ser191Glu + Phe192ValSer190Asp + Ser191Glu + Phe192SerSer190Asp + Ser191Asp + Phe192ThrSer190Glu + Ser191Asp + Asn194SerSer190Glu + Ser191Asp + Phe192AsnSer190Asp + Ser191Asp + Phe192AsnSer190Asp + Ser191Asp + Phe192ValSer190Glu + Ser191Glu + Phe192CysSer190Asp + Ser191Glu + Phe192AsnSer190Glu + Ser191Glu + Phe192LeuSer190Glu + Ser191Asp + Phe192GlySer190Glu + Ser191Glu + Phe192SerSer190Glu + Ser191Glu + Asn194SerSer190Asp + Ser191Asp + Asn194GlnSer190Asp + Ser191Asp + Phe192TyrSer190Asp + Ser191Glu + Phe192MetSer190Asp + Ser191Asp + Phe192GlySer190Glu + Ser191Asp + Asn194GlnSer190Asp + Ser191Glu + Phe192LeuSer190Asp + Ser191Glu + Phe192GlySer190Glu + Ser191Asp + Phe192TyrSer190Asp + Ser191Glu + Phe192ThrSer190Glu + Serl91Asp + Phe192HisSer190Asp + Ser191Asp + Phe192GlnSer190Glu + Ser191Asp + Phe192IleSer190Asp + Ser191Glu + Asn194GlnSer190Asp + Ser191Asp + Phe192ProSer190Glu + Ser191Asp + Phe192CysSer190Asp + Ser191Asp + Asn194SerSer190Asp + Ser191Glu + Phe192G1nSer190Glu + Ser191Asp + Phe192ProSer191Asp + Phe192Asp + Asn194GlnSer191Asp + Phe192Glu + Asn194SerSer191Asp + Phe192Glu + Asn194GlnSer190Asp + Ser191Glu + Phe192AspSer190Asp + Ser191Asp + Phe192GluSer190Glu + Ser191Glu + Phe192AspSer190Glu + Ser191Asp + Phe192AspSer190Glu + Ser191Asp + Phe192Glu
表21
环区5-四突变变异体Ser190Glu + Ser191Asp + Phe192Ile + Asn194SerSer190Asp + Ser191Asp + Phe192Gly + Asn194GlnSer190Glu + Ser191Asp + Phe192His + Asn194SerSerl90G1u + Ser191Asp + Phe192Gln + Asn194GlnSer190Asp + Ser191Asp + Phe192Met + Asn194SerSer190Asp + Ser191Glu + Phe192Leu + Asn194GlnSer190Glu + Ser191Glu + Phe192Ala + Asn194SerSer190Asp + Ser191Asp + Phe192Pro + Asn194GlnSer190Glu + Ser191Glu + Phe192Leu + Asn194SerSer190Glu + Ser191Asp + Phe192His + Asn194GlnSer190Glu + Ser191Asp + Phe192Cys + Asn194GlnSer190Glu + Ser191Asp + Phe192Thr + Asn194SerSer190Glu + Ser191Asp + Phe192Ser + Asn194GlnSer190Glu + Ser191Asp + Phe192Thr + Asn194GlnSer190Asp + Ser191Glu + Phe192Ile + Asn194SerSer190Glu + Ser191Asp + Phe192Gln + Asn194SerSer190Asp + Ser191Asp + Phe192Cys + Asn194GlnSer190Asp + Ser191Asp + Phe192Pro + Asn194SerSer190Glu + Ser191Glu + Phe192Ala + Asn194GlnSer190Asp + Ser191Glu + Phe192Thr + Asn194SerSer190Glu + Ser191Asp + Phe192Leu + Asn194GlnSer190Glu + Ser191Asp + Phe192Gly + Asn194GlnSer190Asp + Ser191Asp + Phe192Cys + Asn194SerSer190Glu + Ser191Glu + Pne192His + Asn194GlnSer190Glu + Ser191Asp + Phe192Asn + Asn194SerSer190Asp + Ser191Asp + Phe192His + Asn194GlnSer190Glu + Ser191Glu + Phe192Ile + Asn194GlnSer190Asp + Ser191Asp + Phe192Tyr + Asn194SerSer190Glu + Ser191Asp + Phe192Tyr + Asn194GlnSer190Glu + Ser191Asp + Phe192Ala + Asn194SerSer190Asp + Ser191Glu + Phe192Ala + Asn194SerSer190Asp + Ser191Glu + Phe192Asn + Asn194SerSer190Glu + Ser191Asp + Phe192Val + Asn194SerSer190Asp + Ser191Asp + Phe192Gln + Asn194SerSer190Asp + Ser191Asp + Phe192Gln + Asn194GlnSer190Asp + Ser191Asp + Phe192Leu + Asn194GlnSer190Asp + Ser191Asp + Phe192Ser + Asn194GlnSer190Glu + Ser191Asp + Phe192Glu + Asn194SerSer190Asp + Ser191Asp + Phe192Asp + Asn194SerSer190Glu + Ser191Asp + Phe192Glu + Asn194GlnSer190Glu + Ser191Glu + Phe192Glu + Asn194SerSer190Asp + Ser191Glu + Phe192Glu + Asn194GlnSer190Asp + Ser191Asp + Phe192Glu + Asn194SerSer190Glu + Ser191Glu + Phe192Asp + Asn194SerSer190Glu + Ser191Asp + Phe192Asp + Asn194SerSer190Glu + Ser191Glu + Phe192Asp + Asn194GlnSer190Asp + Ser191Asp + Phe192Asp + Asn194GlnSer190Asp + Ser191Glu + Phe192Glu + Asn194SerSer190Glu + Ser191Glu + Phe192Tyr + Asn194GluSer190Glu + Ser191Glu + Phe192Met + Asn194GluSer190Asp + Ser191Glu + Phe192Gln + Asn194AspSer190Asp + Ser191Asp + Phe192Ala + Asn194AspSer190Glu + Ser191Glu + Phe192Leu + Asn194AspSer190Glu + Ser191Glu + Phe192Ser + Asn194AspSer190Glu + Ser191Glu + Phe192Met + Asn194AspSer190Asp + Ser191Glu + Phe192Tyr + Asn194GluSer190Asp + Ser191Asp + Phe192His + Asn194AspSer190Glu + Ser191Asp + Phe192Gln + Asn194AspSer190Asp + Ser191Asp + Phe192Asn + Asn194GluSer190Asp + Ser191Glu + Phe192Ile + Asn194Asp
表22环区5-单突变变异体Gly203AsnGly203AspGly203GlnGly203GluGly203ProGly203SerPro204AsnPro204AspPro204GlnPro204GluPro204GlyPro204SerGly205AsnGly205AspGly205GlnGly205GluGly205ProGly205SerThr206AsnThr206AspThr206GlnThr206GluThr206GlyThr206ProThr206SerSer207AspSer207GluIle208AlaIle208AsnIle208AspIle208CysIle208GlnIle208GluIle208GlyIle208HisIle208LeuIle208MetIle208ProIle208SerIle208ThrIle206ValLeu209AlaLeu209AsnLeu209AspLeu209CysLeu209GlnLeu209GluLeu209GlyLeu209HisLeu209IleLeu209MetLeu209ProLeu209SerLeu209ThrLeu209ValSer210AspSer210GluThr211AsnThr211AspThr211GlnThr211GluThr211GlyThr211ProThr211SerTrp212AlaTrp212AsnTrp212AspTrp212CysTrp212GlnTrp212GluTrp212GlyTrp212HisTrp212IleTrp212LeuTrp212MetTrp212PheTrp212ProTrp212SerTrp212ThrTrp212TyrTrp212ValIle213AlaIle213AsnIle213AspIle213CysIle213GlnIle213GluIle213GlyIle213HisIle213LeuIle213MetIle213ProIle213SerIle213ThrIle213ValGly214AsnGly214AspGly214GlnGly214GluGly214ProGly214SerGly215AsnGly215AspGly215GlnGly215GluGly215ProGly215SerSer216AspSer216GluThr217AsnThr217AspThr217GlnThr217GluThr217GlyThr217ProThr217SerArg218AspArg218GluSer219AspSer219GluIle220AlaIle220AsnIle220AspIle220CysIle220GlnIle220GluIle220GlyIle220HisIle220LeuIle220MetIle220ProIle220SerIle220ThrIle220ValSer221AspSer221GluGly222AsnGly222AspGly222GlnGly222GluGly222ProGly222SerThr223AsnThr223AspThr223GlnThr223GluThr223GlyThr223ProThr223Ser
表23环区6-双突变变异体Gly203Gln + Ser219GluThr206Gly + Leu209SerTrp212Phe + Ser221GluArg218Glu + Gly222ProGly214Asp + Ile220SerGly215Asp + Thr217GlyGly203Asn + Ser219AspTrp212Ile + Ser221GluTrp212Gln + Ser219AspTrp212Cys + Ser219AspThr217Glu + Thr223ProGly214Pro + Ile220ValIle208Pro + Thr223SerLeu209Gln + Ile220GluThr206Asn + Ile220HisIle208Ala + Thr217SerPro204Gln + Ser221AspTrp212Thr + Thr223AsnGly203Asn + Ser219GluGly203Gln + Gly214ProGly214Asp + Gly215GlnGly203Ser + Arg218GluThr206Gln + Trp212AsnPro204Ser + Ile213GluIle208Leu + Gly222GluGly215Gln + Thr217GlnIle208Thr + Ser219AspGly205Ser + Gly214AspIle208Ser + Arg218GluIle208Ser + Ile220ValThr211Gly + Arg218AspIle213Asp + Ile220SerPro204Gln + Ser219GluThr211Gly + Ile220MetIle208His + Leu209AlaGly205Asn + Ser207GluThr206Ser + Gly215AsnGly205Asn + Gly222SerGly203Pro + Ile213CysThr206Gln + Gly215SerIle208Ser + Gly214GluThr217Gln + Ser219GluPro204Ser + Ser216AspGly214Asn + Ser216AspIle208Leu + Thr211AsnPro204Gly + Ser207GluSer219Asp + Gly222AsnTrp212Tyr + Ile213ThrThr206Ser + Trp212ValIle220Asp + Thr223ProIle208Pro + Trp212ProIle208Leu + Thr217AspPro204Ser + Gly214SerGly203Ser + Gly222ProGly205Ser + Arg218GluGly203Pro + Ser207AspIle208Val + Ser219GluSer216Asp + Gly222GlnSer207Asp + Trp212AlaIle220Cys + Gly222GluThr206Asn + Ile213CysThr206Asp + Gly215GlnSer207Asp + Thr211SerIle208Leu + Ser216GluLeu209Val + Gly214AspThr206Glu + Ile213AsnPro204Ser + Thr211SerIle213Asp + Gly215ProGly214Asp + Thr223SerGly205Asn + Leu209CysGly205Gln + Ile208AlaIle208Leu + Ser219AspLeu209Glu + Thr211ProIle208Cys + Thr217GlnPro204Asn + Leu209AsnGly214Pro + Ile220ProGly203Gln + Thr211GlyPro204Gly + Thr223AspGly215Ser + Thr217ProThr206Gln + Thr211AsnGly205Pro + Gly215ProGly203Pro + Ser207GluSer207Glu + Ile208HisPro204Ser + Thr223GlyGly205Asn + Gly215AspLeu209Gln + Ser219GluGly215Pro + Thr223SerIle208Gln + Ser221AspGly203Gln + Arg218AspGly203Asn + Ile220AspGly205Pro + Arg218AspThr211Asn + Arg218GluGly205Asn + Thr223GluThr206Glu + Leu209HisThr206Pro + Gly215AsPTrp212Gln + Ser221AspSer216Glu + Ile220MetGly215Asn + Gly222AspIle213Cys + Ser219AspPro204Ser + Thr211AsnPro204Ser + Thr223ProPro204Gln + Ile213GluGly214Ser + Ser221AspIle208Leu + Thr223ProSer207Asp + Ile208GlyIle208Leu + Arg218GluLeu209Ser + Ile220HisIle213Leu + Arg218AspPro204Ser + Ser219AspGly203Ser + Gly222GluGly205Pro + Thr223AsnTrp212Thr + Gly214SerThr206Ser + Ser216AspGly203Pro + Thr206GlyThr211Pro + Gly222SerIle213Thr + Thr223GluThr206Pro + Ile213GluSer207Asp + Gly222AsnIle213Val + Gly214AsnThr211Asn + Thr223AsnIle213Cys + Thr223ProPro204Ser + Gly222GlnGly205Gln + Gly215GluLeu209Pro + Gly222GlnGly205Ser + Gly222AspPro204Ser + Ile208LeuPro204Gln + Thr211GlyLeu209Ser + Ser219AspThr217Glu + Thr223AsnGly203Gln + Thr217SerGly205Pro + Ile213GlnLeu209Glu + Thr217SerSer216Asp + Thr217SerTrp212Gln + Ile213AlaArg218Glu + Ile220GlnLeu209Ile + Thr223AsnIle208Met + Gly215GlnArg218Asp + Ile220AlaGly203Asn + Pro204AsnIle208His + Ser221AspIle213Asn + Ser219AspTrp212Met + Ser219GluThr206Pro + Ser216GluPro204Gln + Thr206AspThr206Gly + Gly215ProPro204Asn + Gly222GlnSer219Glu + Gly222SerIle220Asn + Gly222AsnGly205Asn + Ile213AsnGly203Ser + Ile213LeuSer207Asp + Thr211AsnLeu209Pro + Ile213GluIle208Pro + Thr223AspThr211Ser + Gly222AspThr206Ser + Thr217AspGly203Gln + Gly214GluGly205Ser + Gly215SerGly203Gln + Leu209AlaGly203Gln + Gly222AsnSer207Asp + Thr211GlyGly205Gln + Ser219GluTrp212Cys + Ser216AspGly203Gln + Thr217GluArg218Glu + Ile220CysThr211Gly + Ser221GluPro204Asn + Ser216AspArg218Asp + Gly222ProGly205Pro + Ser221GluArg218Glu + Thr223SerLeu209Asn + Ser216GluGly205Pro + Ile220MetThr206Asn + Thr217AspGly203Ser + Ile208ThrThr217Ser + Arg218GluPro204Asn + Thr211AsnGly205Asn + Thr217GluSer207Asp + Thr223GlyTrp212Tyr + Ile213ProGly214Gln + Ser216AspThr206Asp + Ile208Thr
表24
环区6-三突变变异体Gly203Gln + Thr206Gly + Ser219GluIle213Ala + Gly215Asp + Thr217GlyGly203Asn + Ser219Asp + Thr223SerGly215Pro + Ser216Glu + Thr223SerGly203Gln + Trp212Pro + Gly222AspPro204Ser + Gly205Ser + Ser207GluGly205Pro + Ile208Met + Ser219AspThr211Gly + Gly215Gln + Thr217AsnThr211Ser + Thr217Gln + Thr223AspGly205Gln + Thr211Asn + Gly222AsnThr211Pro + Gly214Asn + Thr217AsnGly205Pro + Leu209Asn + Thr223GlnGly203Gln + Trp212Leu + Ile220AlaTrp212Thr + Gly215Glu + Ile220ThrGly205Gln + Ser207Asp + Trp212AlaGly205Asn + Trp212Phe + Ser216GluSer207Glu + Thr211Gly + Gly222AsnPro204Gln + Gly205Ser + Ser221GluThr206Pro + Leu209Gly + Thr223AsnIle208Met + Thr217Gly + Thr223GlnThr211Ser + Gly215Pro + Gly222GluIle208Pro + Trp212Pro + Ser216GluGly203Ser + Thr211Gln + Gly222ProGly203Pro + Ile208Val + Ser219GluThr206Pro + Gly215Ser + Ser216GluThr206Pro + Ile220Asn + Thr223GlyPro204Gln + Gly205Pro + Arg218GluTrp212His + Ile213Met + Gly215AspIle208Ser + Gly222Gln + Thr223GlnGly205Gln + Trp212Leu + Arg218AspGly214Gln + Ser216Glu + Gly222SerGly203Asn + Ser216Glu + Gly222GlnLeu209Asn + Gly222Glu + Thr223SerThr206Asp + Thr211Asn + Gly214SerPro204Asn + Thr211Gly + Trp212ThrGly203Ser + Ile208Ala + Ser216GluThr211Ser + Gly214Pro + Gly222GluGly215Gln + Arg218Asp + Ile220ValGly215Ser + Ser216Asp + Thr223SerThr206Asn + Thr211Asn + Arg218GluPro204Asn + Thr217Glu + Thr223SerThr206Ser + Ile213Met + Gly215SerPro204Gly + Gly205Ser + Trp212GlnThr211Pro + Trp212Ala + Thr223AspGly215Glu + Ile220Ala + Thr223GlnThr206Gln + Ile208Ser + Leu209AspThr211Ser + Trp212Val + Ser219GluGly205Pro + Thr211Pro + Ser216AspGly205Pro + Trp212His + Ser219GluGly205Pro + Trp212Asn + Ser219AspLeu209Ser + Thr211Gln + Arg218AspPro204Gly + Thr211Asn + Gly215GluGly203Pro + Pro204Ser + Ser216AspGly203Gln + Ile213Val + Arg218GluIle208Gly + Thr217Asn + Gly222SerPro204Ser + Gly205Gln + Trp212IlePro204Gln + Gly214Glu + Gly215GlnTrp212Cys + Gly215Gln + Ser221GluGly203Asn + Leu209Val + Thr217GluGly205Asn + Ser207Asp + Thr217GlyPro204Gln + Thr206Asp + Gly215SerIle208Gln + Thr211Pro + Ser219AspGly205Ser + Ile213His + Ser219GluGly203Ser + Ile208Ser + Trp212MetThr211Pro + Trp212Tyr + Ser219AspGly205Gln + Ile208His + Gly222AsnGly203Ser + Thr211Ser + Ile220GluPro204Gly + Leu209Ile + Arg218GluTrp212Tyr + Ile213Ser + Thr217ProIle208Asn + Ile213Pro + Gly214SerGly203Gln + Ile213His + Ser219AspGly205Asn + Thr211Gln + Ser221GluGly205Asn + Ser207Glu + Gly214GlnIle208Val + Gly214Glu + Gly222GlnGly203Ser + Thr206Gly + Ile213LeuPro204Gly + Ile208Pro + Gly215AspThr206Glu + Thr217Gly + Ile220ProIle213Ser + Thr217Asp + Gly222GlnIle208Val + Gly215Asn + Thr223SerGly205Pro + Ser207Glu + Thr217SerGly203Pro + Thr206Glu + Ile208HisGly205Ser + Trp212Cys + Ser216AspPro204Ser + Thr206Asp + Leu209IleThr206Glu + Ile208Thr + Ile220GlyIle213Gln + Ile220Glu + Gly222GlnIle208Val + Gly214Gln + Gly215ProLeu209Pro + Ser216Glu + Thr217AsnPro204Gln + Trp212Met + Gly222GluIle208Met + Thr211Asn + Thr223GlnPro204Gln + Leu209Ile + Arg218AspIle208Leu + Ser216Glu + Ile220ProPro204Gly + Gly205Gln + Thr206GluIle208Ser + Leu209His + Gly214ProGly203Pro + Ile208Gln + Trp212SerGly205Asn + Gly215Glu + Gly222ProGly203Ser + Thr217Gln + Ile220LeuLeu209Ser + Gly214Ser + Gly222GlnIle208Gly + Leu209Pro + Ser221AspGly205Asn + Thr206Ser + Ile220GlySer207Glu + Leu209Met + Gly222AsnGly203Ser + Trp212Gln + Ser216AspGly205Asn + Ile208Cys + Thr211GlnThr211Gly + Trp212Gly + Thr223GluGly205Asn + Gly214Pro + Ser221GluGly205Ser + Trp212Ala + Ser216AspGly203Gln + Arg218Glu + Ile220LeuSer207Asp + Thr211Asn + Thr223GlyTrp212Cys + Gly215Asn + Thr223ProGly205Asn + Gly214Ser + Arg218GluGly214Pro + Thr217Glu + Gly222AsnThr206Gln + Ile208Asn + Thr211SerGly203Gln + Gly214Pro + Ser219GluTrp212His + Ile213Gln + Arg218GluIle208Ala + Ile213Ala + Ser221GluPro204Asn + Ile208Leu + Trp212ProGly205Asn + Ser219Asp + Ile220HisGly205Gln + Ile208Leu + Thr211GlnTrp212Tyr + Ile213Gly + Gly215GlnTrp212Met + Gly215Glu + Gly222AsnThr206Ser + Thr211Ser + Thr217AsnIle208Leu + Ile213Ser + Ile220ProIle208Ser + Gly214Pro + Gly222AsnThr211Ser + Ser216Glu + Thr223ProGly203Ser + Thr217Pro + Thr223GlyGly203Ser + Trp212Ala + Gly214AspGly205Asn + Ile208Val + Ser219AspGly203Ser + Pro204Gln + Ser221AspGly203Pro + Trp212Gly + Gly222ProIle208Gln + Thr217Gly + Arg218GluThr206Gln + Ser207Glu + Thr223GlySer207Asp + Leu209His + Thr211ProIle208Thr + Trp212Val + Ile220MetLeu209Ala + Ser221Asp + Thr223GlnGly203Asn + Thr211Gly + Gly214GluGly214Pro + Gly215Glu + Ile220ProGly205Ser + Gly214Asn + Ser216GluThr206Gln + Ile213Thr + Ser219GluPro204Gly + Thr211Gly + Ser221AspGly203Pro + Gly205Asn + Gly215AspGly203Pro + Thr211Asn + Thr223AsnPro204Gly + Leu209Pro + Gly215GluGly203Pro + Ile213Glu + Ile220GlyPro204Gln + Ile213Met + Ile220ProGly214Pro + Thr217Gln + Gly222GluIle208His + Ser221Glu + Thr223AsnGly203Pro + Gly214Asn + Arg218AspIle213Ala + Ser216Asp + Gly222SerGly203Asn + Thr211Gly + Thr217AsnLeu209Met + Thr211Gln + Ser219AspThr206Ser + Leu209Ile + Thr211GlyIle213Met + Gly215Gln + Ser221AspPro204Asn + Ser207Asp + Ile220HisGly203Gln + Ile208Val + Ser221AspThr217Gln + Ile220Val + Thr223GluGly205Asn + Ile208Val + Ile213HisThr206Asn + Thr211Gly + Thr217AspSer207Glu + Ile208Cys + Gly215ProPro204Asn + Gly205Pro + Trp212IlePro204Gly + Ile208Val + Thr223AspPro204Gly + Gly215Asn + Ile220HisGly205Asn + Thr211Asn + Trp212ProPro204Ser + Thr206Glu + Ile213ValGly203Pro + Ser207Glu + Gly214AsnPro204Gln + Gly214Glu + Gly215GluGly205Ser + Thr217Asp + Arg218AspLeu209Met + Thr217Asp + Arg218AspThr217Asp + Arg218Asp + Ile220CysThr211Gln + Ser221Glu + Gly222AspGly205Pro + Thr206Asp + Ser207GluPro204Gly + Thr206Asp + Ser207GluThr211Gln + Ile220Glu + Ser221AspTrp212Tyr + Arg218Asp + Ser219GluIle208Val + Arg218Glu + Ser219GluGly205Gln + Gly215Asp + Ser216AspLeu209Glu + Trp212Phe + Ser219AspThr206Glu + Ile208Ala + Ser221AspGly205Asn + Ser207Glu + Ser221GluSer207Glu + Thr211Asn + Ile220AspGly203Pro + Ser207Glu + Ile220GluSer207Asp + Trp212His + Ser219Asp
表25
环区6-四突变变异体Gly203Gln + Thr206Gly + Leu209Ser + Ser219GluIle208Val + Thr217Ser + Ser219Asp + Gly222ProGly203Asn + Ile213Thr + Gly214Pro + Ser219GluGly203Gln + Trp212Phe + Gly214Asn + Gly215AspSer207Asp + Leu209Thr + Thr211Pro + Trp212GlyGly205Pro + Trp212Asn + Ile213His + Ile220AspGly203Gln + Pro204Ser + Trp212Ile + Ile220SerPro204Gln + Thr211Gly + Ser219Glu + Ile220MetGly203Gln + Trp212Gly + Gly215Glu + Ile220AlaThr206Gln + Thr211Pro + Gly215Asn + Ile220ThrLeu209Val + Thr211Ser + Arg218Asp + Ile220SerGly203Gln + Ile208Cys + Leu209Ser + Gly214GluGly205Gln + Ser207Glu + Gly215Gln + Ile220AlaIle208Ala + Leu209Val + Ser216Glu + Thr223ProPro204Gly + Ile208His + Ser219Glu + Ile220SerThr206Asp + Ile208Gly + Thr217Asn + Thr223SerGly203Asn + Leu209Ser + Gly214Ser + Ser221AspIle213Asp + Gly214Ser + Ile220Gln + Gly222AsnTrp212Thr + Ile213Gly + Gly214Ser + Ile220AlaGly203Asn + Leu209Asp + Trp212Phe + Gly222GlnThr206Asn + Thr211Ser + Ile213Gly + Ser219AspPro204Asn + Gly205Gln + Thr206Asn + Ser207GluGly203Gln + Gly205Asn + Ser207Asp + Leu209ThrGly205Gln + Ile213Val + Gly214Pro + Arg218GluThr206Pro + Ile213Pro + Gly222Pro + Thr223AspGly203Asn + Gly214Asn + Ser221Asp + Thr223GlyIle213Asn + Ser216Asp + Ile220Asn + Gly222AsnPro204Ser + Leu209Pro + Ile213Glu + Ile220ProGly203Gln + Gly205Ser + Leu209Ala + Gly215SerGly203Gln + Ser207Asp + Thr211Gly + Gly222AsnPro204Gln + Leu209His + Thr217Gly + Ser219GluThr206Asp + Ile208Thr + Leu209Ala + Thr217AsnIle208Thr + Leu209Gly + Arg218Asp + Ile220LeuPro204Asn + Ile208Val + Leu209His + Arg218GluIle208Asn + Thr211Gln + Ser219Glu + Gly222GlnThr206Gly + Ser207Glu + Ile208Thr + Ile213GlyLeu209Glu + Gly214Gln + Ile220Val + Gly222GlnGly205Gln + Gly214Asn + Thr217Gln + Thr223GluGly203Gln + Leu209Val + Gly215Pro + Ile220GluThr211Gln + Trp212Gln + Gly215Asn + Ser219GluPro204Ser + Ile208Ser + Gly222Glu + Thr223GlnPro204Gly + Ile208Ser + Gly215Gln + Ser221AspGly203Gln + Thr211Gly + Ile213Leu + Gly214AsnPro204Asn + Thr211Ser + Gly214Asp + Thr217AsnPro204Gly + Trp212Met + Gly215Glu + Gly222AsnThr206Glu + Ile208Leu + Ile213Ser + Ile220ProThr206Gly + Trp212Leu + Gly214Asp + Gly222SerGly203Ser + Gly214Asp + Thr217Pro + Thr223GlyGly203Ser + Pro204Asn + Ile208Ala + Gly215GluGly203Ser + Thr211Ser + Ser219Glu + Gly222AsnSer207Glu + Ile208Gly + Thr211Pro + Ile220ProGly203Asn + Gly205Pro + Leu209Ala + Gly222GluLeu209Asn + Thr211Gly + Ile213Thr + Thr223GlnGly203Pro + Pro204Asn + Ile213His + Thr217GlnIle208Asn + Thr217Pro + Ile220Leu + Ser221GluThr206Ser + Thr211Gln + Ile213Gly + Ser221AspGly205Pro + Gly215Asp + Ile220His + Thr223GlyPro204Gln + Ile208Thr + Ser219Asp + Gly222AsnGly203Pro + Leu209Met + Thr211Pro + Ile220CysThr206Gly + Leu209Cys + Ile213His + Ser216AspIle208Gly + Leu209Met + Thr217Gly + Ile220ProGly203Asn + Thr206Ser + Gly214Gln + Ser221GluIle208His + Thr211Ser + Trp212Pro + Arg218AspThr206Ser + Ile208Pro + Leu209Ile + Gly214AspGly205Pro + Thr206Glu + Ile213His + Gly222AsnThr211Asn + Ile213Gln + Thr217Asp + Thr223SerThr206Asn + Trp212Val + Arg218Glu + Ile220ProPro204Asn + Ser207Asp + Thr211Gln + Gly214SerPro204Ser + Ile213Met + Gly214Asp + Thr217GlyGly203Pro + Ile208Ala + Ser216Glu + Thr217ProSer207Asp + Trp212Cys + Gly215Pro + Thr217GlyPro204Asn + Gly214Gln + Ser216Glu + Ile220HisPro204Ser + Trp212Ala + Gly214Asp + Gly215ProPro204Asn + Leu209Gly + Thr211Gly + Ile213ThrPro204Ser + Gly205Gln + Ile208His + Ser219AspGly203Asn + Gly205Ser + Leu209Ser + Arg218GluThr206Pro + Thr211Gln + Ile213Glu + Gly214AsnSer207Asp + Gly214Asn + Ile220Val + Gly222AsnTrp212Tyr + Ile213Val + Gly214Asn + Gly222SerGly205Pro + Ile208Gly + Ser216Asp + Ile220HisGly203Asn + Leu209Gly + Gly214Pro + Arg218AspPro204Gln + Ile208Cys + Ile213Gly + Gly215GluPro204Asn + Gly205Pro + Leu209Ala + Thr211ProGly203Pro + Ile213Ala + Thr217Gly + Arg218GluGly203Gln + Pro204Gly + Ile220Gly + Ser221GluGly205Asn + Ile213Gly + Gly214Pro + Gly222SerGly203Asn + Thr211Gln + Gly214Glu + Gly222GlnIle213Thr + Gly215Gln + Ser219Asp + Ile220ProGly203Asn + Pro204Ser + Arg218Asp + Thr223ProGly203Ser + Thr211Gln + Arg218Glu + Ile220ValGly203Ser + Pro204Ser + Ile208Ser + Thr211GlnPro204Gly + Gly214Asn + Gly215Glu + Thr223AsnIle208Cys + Thr211Pro + Trp212Ser + Gly215ProPro204Asn + Ile213Met + Thr217Glu + Ile220GlnPro204Gly + Gly205Pro + Ile208Gly + Thr211ProGly203Ser + Thr206Gly + Ile208Asn + Ser219AspPro204Asn + Ile213Glu + Gly215Pro + Thr217AsnGly203Asn + Thr211Gln + Arg218Glu + Ile220GlyGly203Gln + Ile208Cys + Gly215Ser + Arg218AspPro204Gln + Gly205Gln + Gly214Ser + Thr223GlyPro204Gln + Leu209Met + Trp212Phe + Ser219GluGly205Asn + Ile208Cys + Leu209Ile + Ile220AsnGly203Pro + Leu209Cys + Thr217Gly + Ile220GlyGly205Asn + Thr206Gly + Ile208Asn + Ser216GluPro204Gln + Thr206Pro + Ser207Glu + Gly214AsnGly203Gln + Ile208Met + Trp212His + Thr223ProPro204Asn + Leu209Val + Thr211Asn + Gly215SerGly203Asn + Ile208His + Ser221Asp + Gly222SerPro204Asn + Leu209Pro + Ser219Glu + Ile220GluThr211Pro + Ile213Asn + Gly214Glu + Gly215AspThr211Gln + Gly215Ser + Thr217Asp + Arg218AspGly205Asn + Thr217Asp + Arg218Asp + Ile220GlnIle208Thr + Leu209Thr + Ser221Asp + Gly222GluGly203Asn + Gly205Asn + Ser221Asp + Gly222GluThr206Ser + Trp212Pro + Ser221Asp + Gly222AspIle208Pro + Thr211Ser + Gly222Asp + Thr223AspPro204Ser + Trp212Met + Gly222Glu + Thr223AspGly203Gln + Thr206Glu + Ser207Glu + Thr211GlnGly203Asn + Thr206Glu + Ser207Asp + Thr211AsnThr206Asp + Ser207Asp + Ile220Thr + Thr223ProGly203Gln + Thr217Ser + Ile220Glu + Ser221AspGly205Ser + Thr211Gln + Arg218Glu + Ser219AspLeu209Gly + Gly214Ser + Arg218Glu + Ser219GluIle208Gln + Gly214Pro + Ser216Asp + Thr217GluIle208Gly + Ile213Met + Gly215Glu + Ser216GluIle208Ser + Leu209Asp + Thr211Gly + Ser219AspPro204Gln + Ile208Thr + Leu209Asp + Ser219AspThr206Asp + Trp212Gln + Ser221Asp + Thr223GlyGly203Asn + Thr206Asp + Trp212Phe + Ser221GluPro204Gln + Thr206Glu + Leu209Ile + Ser221GluPro204Gln + Gly205Gln + Ser207Glu + Ser221GluSer207Asp + Ile208Leu + Trp212Ile + Ser221GluSer207Glu + Leu209Met + Ile220Glu + Thr223SerThr217Asn + Arg218Asp + Ser219Asp + Ile220AspPro204Asn + Ser207Asp + Ser219Glu + Gly222GlnPro204Gly + Gly205Pro + Ser207Glu + Ser219GluGly205Gln + Ser207Asp + Trp212Val + Ser219GluSer207Glu + Ile208Ser + Ser219Glu + Thr223GlyThr206Asp + Thr217Ser + Ile220Asp + Ser221GluTrp212Gln + Gly214Asp + Ser216Glu + Ile220ValGly214Asp + Gly215Gln + Ser216Glu + Thr223AsnGly203Gln + Leu209Asp + Thr211Asn + Arg218GluPro204Ser + Leu209Glu + Arg218Asp + Ile220CysSer207Asp + Ile208Gly + Ser221Glu + Gly222AspPro204Asn + Ser207Glu + Ser221Asp + Gly222AspPro204Ser + Gly205Ser + Ile213Asp + Ser216AspThr206Asn + Ser219Glu + Ser221Glu + Thr223AsnThr206Pro + Trp212Pro + Ser216Glu + Arg218GluGly203Ser + Gly215Asn + Ser216Asp + Arg218GluThr206Gln + Trp212Asn + Ser216Asp + Arg218GluLeu209Val + Trp212Ile + Ser216Asp + Arg218GluPro204Gly + Ile208Pro + Ser216Asp + Arg218AspIle208Gly + Thr211Asn + Ser216Glu + Arg218GluTrp212His + Ile213Asn + Thr217Glu + Ser219GluIle208Leu + Thr211Pro + Thr217Asp + Ser219GluGly203Asn + Thr217Asp + Ser219Glu + Ile220AlaThr206Pro + Thr217Glu + Ser219Glu + Ile220AlaGly203Gln + Gly215Glu + Thr217Glu + Ile220CysGly203Asn + Arg218Glu + Ser219Asp + Ser221GluLeu209Met + Ser219Asp + Ser221Glu + Gly222AspThr211Ser + Ile213Glu + Ser216Asp + Arg218AspThr211Asn + Ser216Asp + Arg218Asp + Ser219AspSer216Glu + Arg218Asp + Ser219Glu + Thr223SerGly203Pro + Thr206Glu + Leu209Asp + Ile220AspSer207Glu + Leu209Gln + Gly222Asp + Thr223SerGly205Gln + 5er207Glu + Ile213Gln + Gly222AspGly205Asn + Ser207Glu + Ile213Leu + Gly222AspGly205Gln + Ser207Glu + Gly222Asp + Thr223GluSer207Asp + Trp212Gly + Ser219Glu + Gly222GluTrp212Thr + Gly214Glu + Ser216Asp + Arg218AspSer207Asp + Arg218Asp + Ser221Asp + Gly222AsnSer207Asp + Trp212His + Arg218Glu + Ser221GluPro204Asn + Thr206Asp + Thr211Ser + Ser219AspThr206Asn + Ile213Pro + Gly214Glu + Thr217AspThr211Gln + Gly214Glu + Gly215Ser + Thr217GluLeu209Asp + Gly214Pro + Ser221Glu + Gly222AspSer207Glu + Leu209Ala + Ile213Ala + Arg218GluPro204Gln + Ser207Glu + Trp212Leu + Arg218AspPro204Gly + Ile213Glu + Arg218Glu + Ser219GluPro204Gly + Thr206Glu + Leu209Asp + Thr223Gly
表26
环区6-五替换突变体Pro204Gln + Gly205Gln + Ile208Ala + Ser216Asp +
Gly222ProPro204Gln + Ser207Glu + Ile208Ser + Thr211Gly +
Ile220AlaGly205Gln + Trp212Phe + Gly214Pro + Gly215Asp +
Thr217GlyGly203Asn + Ile208Gln + Leu209Gln + Thr211Pro +
Thr223AspGly203Ser + Ile208Cys + Gly214Gln + Ser219Glu +
Ile220ThrTrp212Gln + Ile213Ser + Gly214Asn + Thr217Glu +
Ile220SerGly205Ser + Ile208Pro + Trp212Tyr + Gly214Gln +
Ser221AspGly205Gln + Thr206Glu + Ile208Thr + Leu209Thr +
Trp212CysGly203Asn + Gly205Pro + Ile208Val + Gly214Asn +
Ser219GluGly203Gln + Gly205Gln + Ile208Gly + Thr217Asn +
Gly222SerIle208Met + Thr211Gly + Ile213Cys + Gly214Pro +
Ile220LeuThr211Pro + Ile213Ser + Thr217Asp + Ile220Pro +
Gly222GlnPro204Asn + Gly205Ser + Thr211Gly + Ile220Thr +
Gly222AsnGly203Asn + Gly205Asn + Leu209His + Ile220His +
Thr223GlnPro204Gly + Ile208Asn + Thr217Gln + Gly222Glu +
Thr223GlnGly205Gln + Ser207Asp + Ile208Met + Ile213Cys +
Gly214ProGly205Asn + Thr211Gln + Trp212Met + Thr217Glu +
Gly222AsnGly205Gln + Thr206Pro + Leu209Glu + Ile213Thr +
Ile220GlyThr211Asn + Gly214Gln + Gly215Ser + Thr217Ser +
Gly222SerPro204Gly + Gly205Gln + Thr211Gly + Ser221Glu +
Thr223GlnGly203Ser + Ile208Thr + Ile220Val + Gly222Pro +
Thr223GluGly205Asn + Thr206Pro + Ser216Glu + Thr217Gln +
Gly222SerGly203Asn + Pro204Gln + Ile208Met + Gly215Gln +
Ser219AspGly205Gln + Thr206Pro + Trp212Ser + Ile213Gln +
Ser221GluLeu209Gln + Gly214Gln + Thr217Ser + Ser221Asp +
Thr223AsnSer207Glu + Trp212Tyr + Ile213Gly + Gly214Pro +
Ile220ProGly203Pro + Trp212Pro + Gly214Glu + Gly215Pro +
Gly222ProPro204Ser + Thr206Asn + Thr211Gly + Trp212Gln +
Thr223GlnPro204Gly + Thr206Pro + Trp212Val + Gly214Pro +
Arg218GluGly203Gln + Thr211Gln + Gly215Aan + Thr217Glu +
Ile220ValGly203Gln + Gly205Pro + Leu209Val + Thr211Gln +
Gly214AspPro204Asn + Ile213Thr + Gly215Gln + Ser219Asp +
Ile220ProPro204Gln + Thr206Asn + Ile208Met + Gly215Glu +
Ile220AlaGly203Ser + Gly205Pro + Ile208Asn + Leu209Glu +
Thr211GlnPro204Gly + Gly205Gln + Thr211Ser + Trp212Met +
Thr217ProGly205Ser + Thr206Gly + Ile208Val + Gly214Asp +
Thr223ProSer207Glu + Ile208Ala + Gly215Pro + Thr217Gln +
Thr223ProGly203Ser + Gly205Gln + Trp212Asn + Ile213Ser +
Thr223ProPro204Gln + Ile208Ala + Leu209Gln + Thr211Ser +
Gly214SerPro204Gln + Ile208His + Thr211Gln + Gly215Pro +
Ser216AspPro204Gln + Leu209Ile + Thr211Ser + Gly214Asp +
Ile220CysLeu209Ile + Thr211Gln + Trp212Asn + Ile213Val +
Thr217AspPro204Asn + Ile208His + Leu209Val + Thr211Asn +
Gly222SerGly203Gln + Pro204Asn + Gly205Asn + Ile213Ala +
Gly214AspSer207Asp + Thr211Gln + Trp212Gly + Gly222Gln +
Thr223GlyGly205Gln + Ile208Gly + Thr211Gln + Ile220Ser +
Gly222AspGly205Pro + Ile208Pro + Leu209Val + Ile220Leu +
Gly222GluPro204Gly + Thr206Glu + Leu209Cys + Ile213His +
Ile220MetLeu209Gly + Trp212Val + Ser216Asp + Ile220Cys +
Thr223SerGly205Gln + Leu209Val + Thr211Asn + Gly214Pro +
Gly222AspIle208Ala + Leu209His + Gly215Asn + Ile220Val +
Gly222GluSer207Glu + Ile208Pro + Thr211Asn + Ile213His +
Gly222SerIle208His + Leu209Cys + Thr217Pro + Ile220Glu +
Gly222GlnPro204Gly + Gly205Ser + Ile208Ala + Trp212Leu +
Thr223AsnGly203Pro + Gly205Pro + Trp212Ala + Gly215Ser +
Arg218GluPro204Ser + Trp212Ala + Ile213Thr + Gly215Asn +
Ser216GluThr211Asn + Trp212Thr + ser216Glu + Gly222Gln +
Thr223ProThr206Gln + Ile208Pro + Thr211Gly + Ile213Leu +
Gly215AspGly203Asn + Ile220Met + Ser221Glu + Gly222Gln +
Thr223AsnPro204Gln + Thr206Gly + Trp212Gly + Gly215Asp +
Ile220CysGly203Asn + Pro204Gln + Trp212Pro + Ile220Gly +
Thr223AspPro204Ser + Thr206Ser + Ile208Leu + Thr211Ser +
Gly222ProGly203Ser + Ile208Thr + Leu209His + Thr211Gln +
Ile220GluPro204Asn + Thr211Pro + Gly214Gln + Gly215Glu +
Thr217SerPro204Asn + Thr211Pro + Thr217Asp + Ile220Val +
Thr223GlnGly203Ser + Ile208Gln + Leu209Asn + Thr211Ser +
Trp212AsnPro204Asn + Ile208His + Thr211Gln + Ile220Gly +
Gly222AspGly205Pro + Trp212Val + Gly215Pro + Ser216Asp +
Thr217ProGly203Gln + Ile208Ala + Thr211Pro + Gly214Ser +
Arg218GluGly203Asn + Pro204Gln + Leu209Met + Trp212Ser +
Thr217GluGly205Pro + Ser207Glu + Ile208Met + Leu209Met +
Ile220AsnThr211Gly + Trp212Asn + Ser219Glu + Ile220Asp +
Thr223ProIle208Met + Ile213Thr + Thr217Asp + Arg218Glu +
Thr223GlyGly203Ser + Ile208Gln + Thr217Glu + Arg218Asp +
Gly222ProGly205Gln + Thr217Glu + Arg218Glu + Ile220His +
Thr223ProGly203Ser + Ile213Gln + Thr217Asp + Arg218Asp +
Gly222GlnGly203Pro + Pro204Gly + Ile208Gly + Ser221Asp +
Gly222AspGly203Asn + Thr211Gln + Ile213Asp + Gly214Glu +
Gly222GlnThr206Glu + Ser207Glu + Leu209Asn + Thr211Gln +
Thr217AsnGly203Gln + Leu209Met + Thr211Pro + Ile220Glu +
Ser221GluIle208Val + Leu209His + Thr211Gln + Arg218Glu +
Ser219GluGly203Pro + Pro204Asn + Leu209His + Arg218Asp +
Ser219AspIle208Ala + Trp212Gln + Arg218Glu + Ser219Glu +
Ile220ThrLeu209Gly + Thr211Pro + Gly215Asn + Ser216Glu +
Thr217AspIle208Asn + Leu209Thr + Ile213His + Ser216Glu +
Thr217GluLeu209Asn + Gly215Glu + Ser216Asp + Ile220Asn +
Thr223AsnGly203Ser + Pro204Gly + Gly215Glu + Ser216Asp +
Thr223SerPro204Gln + Ile208Cys + Ile213Cys + Gly215Glu +
Ser216GluPro204Gln + Gly205Asn + Thr206Asp + Ser207Glu +
Ser221GluGly203Gln + Pro204Asn + Thr206Asp + Ile208Cys +
Ser221GluPro204Gly + Thr206Glu + Ile208Gln + Trp212His +
Ser221GluThr206Asp + Ile208Met + Trp212Met + Ile213Met +
Ser221GluSer207Asp + Thr211Gln + Trp212Ala + Ile213Pro +
Ser221AspThr206Gln + Ser207Asp + Leu209Val + Thr211Gly +
Ser221AspPro204Ser + Ser207Glu + Ile208Thr + Trp212Ile +
Ser221AspSer207Glu + Leu209Ala + Thr217Gly + Ile220Ala +
Ser221AspSer207Asp + Thr211Pro + Trp212Cys + Gly215Pro +
Ser221GluGly205Pro + Ser207Glu + Trp212Asn + Ser221Glu +
Thr223SerGly205Gln + Ser207Glu + Thr211Gly + Thr217Gly +
Ser221GluThr206Gln + Ser207Glu + Ile208Ala + Leu209Cys +
Ile220GluThr211Gln + Arg218Glu + Ser219Asp + Ile220Asp +
Gly222SerGly205Asn + Thr206Pro + Ser207Asp + Ser219Glu +
Gly222GlnPro204Ser + Ser207Asp + Ser219Asp + Ile220Met +
Thr223ProThr206Asn + Ser207Glu + Ile208Thr + Ile213Gly +
Ser219GluGly205Gln + Leu209Asn + Thr217Asp + Arg218Glu +
Ser219AspPro204Gly + Ile208Val + Ile213Asp + Gly214Glu +
Ser216AspPro204Ser + Ile220His + Ser221Glu + Gly222Glu +
Thr223GluThr206Asp + Trp212Val + Ile220Glu + Ser221Asp +
Thr223ProThr206Glu + Ser207Asp + Gly214Asn + Ile220Asp +
Thr223AsnIle208Leu + Leu209Val + Thr211Ser + Gly214Asp +
Ser216GluThr211Ser + Ile213His + Gly214Asp + Ser216Asp +
Ile220GlnGly205Pro + Thr206Ser + Leu209Glu + Thr217Asn +
Arg218GluGly205Ser + Leu209Asp + Gly214Ser + Gly215Ser +
Arg218AspSer207Glu + Leu209Gly + Trp212Phe + Ser219Glu +
Ser221GluGly203Pro + Pro204Asn + Thr211Ser + Ile213Glu +
Ser216GluThr206Glu + Ser207Asp + Ile208Ala + Gly215Pro +
Ser219GluThr206Glu + Leu209Ser + Trp212Ile + Ile220Thr +
Gly222GluThr206Asp + Ser207Asp + Ile208Leu + Ile213Pro +
Gly222GluGly203Ser + Thr206Asp + Ser207Glu + Ile220Gln +
Gly222GluThr211Gly + Ile213Ala + Gly214Glu + Ser216Glu +
Thr217AspSer207Asp + Thr211Gln + Arg218Glu + Ser219Glu +
Ile220ValSer207Glu + Leu209Glu + Gly214Gln + Ile220Val +
Gly222GlnSer207Glu + Leu209Glu + Thr211Gln + Gly214Gln +
Thr217GlnGly205Gln + Ser207Glu + Thr211Ser + Arg218Asp +
Ile220GluLeu209Glu + Thr217Pro + Ser219Asp + Ser221Glu +
Gly222SerThr211Gly + Gly214Asn + Ser219Glu + Ile220Leu +
Ser221AspGly203Pro + Ile208Gln + Gly214Pro + Ser219Asp +
Ser221GluPro204Asn + Leu209Ile + Ser219Glu + Ser221Asp +
Gly222SerThr211Ser + Trp212Gln + Gly214Gln + Ser219Asp +
Ser221AspLeu209Ala + Gly214Pro + Ser219Glu + Ile220Met +
Ser221GluPro204Gly + Gly205Gln + Ile213Val + Ser216Asp +
Arg218GluPro204Gly + Thr206Pro + Thr211Asn + Ser216Glu +
Arg218GluThr206Asp + Ile208Thr + Leu209Ala + Thr217Asn +
Ile220AspPro204Gln + Thr206Glu + Ile208Pro + Trp212His +
Ile220GluThr206Gln + Thr211Ser + Thr217Asp + Ser219Asp +
Thr223ProGly203Ser + Pro204Ser + Gly205Pro + Gly215Asp +
Thr217GluGly203Pro + Pro204Gln + Gly215Glu + Thr217Glu +
Gly222GlnPro204Ser + Leu209Met + Gly215Glu + Thr217Asp +
Thr223AsnPro204Gly + Trp212Leu + Gly214Ser + Ser221Asp +
Thr223AspLeu209Ser + Thr211Asn + Arg218Asp + Ile220Glu +
Ser221GluIle208Gly + Leu209Asn + Ser219Asp + Ser221Glu +
Gly222AspPro204Gly + Ile208Leu + Gly215Asp + Ser216Glu +
Arg218AspPro204Gln + Ile208Gly + Gly215Asp + Ser216Glu +
Arg218GluThr206Gly + Ile213Ser + Ser216Glu + Arg218Asp +
Ser219GluPro204Gln + Thr206Glu + Ser207Asp + Thr211Asn +
Thr223GluPro204Gly + Gly205Ser + Ser207Glu + Arg218Asp +
Ser221AspThr206Asp + Ile213Thr + Gly214Ser + Ser219Asp +
Gly222GluGly203Asn + Thr206Asn + Ile213Pro + Gly214Glu +
Thr217AspPro204Ser + Ile213Gly + Gly214Glu + Thr217Asp +
Ile220AlaSer207Glu + Thr217Asp + Arg218Asp + Ile220Ala +
Thr223SerSer207Asp + Leu209Ser + Trp212Val + Gly215Ser +
Arg218GluPro204Asn + Ser207Glu + Leu209Val + Trp212Phe +
Arg218AspGly205Ser + Ser207Glu + Gly215Gln + Arg218Glu +
Thr223AsnGly205Ser + Leu209Asp + Ile213Pro + Gly215Asn +
Ser221AspGly205Pro + Ser207Glu + Leu209Glu + Thr217Ser +
Gly222AspSer207Asp + Leu209Asp + Thr217Gly + Gly222Asp +
Thr223SerGly203Asn + Ile208Ser + Leu209Asp + Ser216Glu +
Ser219AspLeu209Ala + Trp212Ser + Gly214Glu + Gly215Glu +
Arg218GluGly205Gln + Thr206Glu + Ile213Gln + Arg218Glu +
Ser221GluPro204Gly + Leu209Asn + Gly215Pro + Thr217Asp +
Ile220AspThr206Pro + Trp212Ala + Arg218Asp + Ser219Glu +
Gly222AspGly203Asn + Gly205Pro + Trp212Asn + Arg218Glu +
Ser221AspThr206Gly + Ile208Ala + Trp212Tyr + Arg218Asp +
Ser221GluGly205Gln + Thr211Ser + Arg218Asp + Ser221Glu +
Thr223ProGly205Pro + Thr211Pro + Trp212Pro + Arg218Glu +
Ser221GluGly203Ser + Arg218Asp + Ile220Asn + Ser221Asp +
Thr223SerTrp212His + Ile213Asn + Gly214Glu + Thr217Glu +
Ser219GluGly205Pro + Ile213Pro + Gly215Asp + Arg218Glu +
Ser219AspPro204Ser + Gly215Asp + Arg218Glu + Ser219Glu +
Gly222AsnSer207Glu + Gly214Gln + Ser216Asp + Arg218Glu +
Gly222GlnSer207Asp + Gly215Pro + Ser216Glu + Thr217Asn +
Arg218AspGly205Ser + Ile208Cys + Leu20gAla + Gly215Asp +
Arg218AspGly203Asn + Ser207Asp + Ser216Glu + Ser219Asp +
Gly222ProSer207Glu + Leu209Ile + Ser216Glu + Thr217Gly +
Ser219GluLeu209Asp + Trp212Pro + Gly214Glu + Ser216Glu +
Ile220CysGly203Gln + Thr206Gly + Leu209Ser + Ser216Glu +
Ser219GluIle208Leu + Gly214Asn + Ser216Glu + Thr217Ser +
Ser219GluLeu209Ser + Ser216Glu + Thr217Ser + Ser219Glu +
Ile220HisPro204Gln + Leu209Pro + Gly214Ser + Ser216Glu +
Ser219AspThr206Gly + Ile208Met + Gly215Asp + Arg218Asp +
Ile220Glu
表27
环区6-六替换变异体Gly203Asn + Thr206Asn + Ile208Asn + Trp212Asn +
Ile213Leu + Ser221GluPro204Gln + Gly205Gln + Ile208Ala + Leu209Pro +
Ser216Asp + Gly222ProGly203Gln + Gly205Asn + Gly215Ser + Thr217Ser +
Ile220Met + Ser221AspIle208Pro + Leu209Ala + Gly215Gln + Arg218Asp +
Ile220Val + Thr223SerGly203Ser + Leu209Cys + Trp212Tyr + Ser216Asp +
Thr217Asn + Ile220ValGly203Gln + Gly205Ser + Leu209Ala + Ile213Met +
Gly215Asn + Gly222AsnGly203Pro + Gly205Ser + Leu209Ala + Trp212Ala +
Gly214Gln + Ser219AspGly203Pro + Leu209Ile + Thr211Pro + Ile213Val +
Gly215Pro + Ile220ProThr206Ser + Ile208Asn + Thr211Gln + Ile213Gly +
Ile220Leu + Ser221AspPro204Ser + Gly205Pro + Ile208Thr + Gly215Asp +
Ile220His + Thr223GlyGly203Pro + Pro204Gln + Leu209Met + Thr211Pro +
Ser219Asp + Ile220CysPro204Asn + Thr206Asn + Ser207Asp + Thr211Gln +
Gly214Ser + Ile220ProGly203Ser + Leu209Pro + Trp212Met + Ile213Cys +
Thr217Pro + Arg218GluPro204Asn + Gly205Ser + Ile208Ala + Ile213Pro +
Gly215Ser + Ile220GlyGly203Ser + Gly205Asn + Ser207Glu + Thr211Ser +
Trp212Val + Thr217AsnGly203Gln + Pro204Asn + Ile208His + Leu209Pro +
Ile213Leu + Gly222AsnGly205Asn + Thr206Gly + Ile208Pro + Thr211Pro +
Trp212Ile + Thr223AsnGly205Gln + Thr206Ser + Ile208Gln + Ser221Asp +
Gly222Gln + Thr223ProIle208Leu + Trp212Asn + Ile213Thr + Gly214Ser +
Thr217Gln + Ser219GluGly203Gln + Pro204Gln + Leu209His + Ser219Glu +
Gly222Asn + Thr223GlnPro204Gln + Gly205Asn + Ile208Cys + Leu209Ile +
Ile220Asn + Thr223GluGly203Pro + Gly205Gln + Thr206Gly + Thr211Gln +
Trp212Pro + Gly215GluThr206Gln + Ile208Leu + Thr211Gln + Gly214Asn +
Ile220Asn + Thr223AsnIle208Val + Leu209Cys + Thr211Ser + Ile213Gly +
Gly214Asp + Thr223GlyPro204Ser + Gly205Asn + Leu209Ser + Thr217Gly +
Arg218Glu + Gly222SerPro204Ser + Gly205Gln + Leu20gHis + Thr211Gln +
Gly214Ser + Ser221GluIle208His + Leu209Cys + Thr211Ser + Trp212Ser +
Gly214Glu + Gly222AsnIle208Gln + Leu209Gly + Trp212Phe + Thr217Pro +
Ile220Thr + Gly222GluGly205Pro + Ser207Glu + Ile208Met + Leu209Met +
Trp212Cys + Ile220AsnPro204Ser + Thr206Asn + Ile208Gln + Trp212Phe +
Ser219Glu + Thr223GlyGly205Pro + Ile208Cys + Trp212Ile + Thr217Gln +
Ile220Leu + Gly222ProPro204Ser + Gly205Ser + Ile208Cys + Ile213Pro +
Ser221Asp + Thr223GlnPro204Ser + Thr206Gln + Trp212Met + Gly214Ser +
Ser221Asp + Thr223SerGly205Ser + Ile208Gln + Ile213Cys + Gly215Pro +
Thr217Gln + Thr223AspPro204Asn + Gly205Pro + Ile208Ser + Ile213Asp +
Ile220Thr + Gly222GlnPro204Asn + Ile208Ala + Leu209Ser + Thr211Gly +
Gly215Ser + Ser219GluGly203Gln + Gly205Gln + Thr206Gln + Ser207Glu +
Ile208Ala + Ile220AlaLeu209Ser + Thr211Gly + Trp212Tyr + Ile213His +
Ser216Asp + Thr223ProPro204Gln + Thr206Ser + Ile208Met + Leu209Cys +
Thr211Ser + Gly214AspGly203Asn + Leu209Gln + Thr211Ser + Gly215Pro +
Ile220Cys + Gly222ProLeu209His + Thr211Asn + Trp212Ile + Ile213Ala +
Ser219Glu + Thr223GlyThr206Gln + Ile208His + Trp212Gln + Gly215Ser +
Ser221Asp + Thr223GlnIle208Cys + Leu209Met + Trp212Ser + Ile213Gln +
Ser216Glu + Thr223ProGly205Ser + Leu209Met + Trp212Gly + Arg218Glu +
Ile220Gly + Gly222AsnPro204Asn + Trp212Gln + Gly215Ser + Ser216Glu +
Thr217Asn + Ile220AsnGly203Pro + Ile208Val + Leu209Asp + Trp212Ala +
Ile220Gln + Thr223GlyGly203Gln + Gly205Pro + Ile208Gly + Trp212Ser +
Thr217Asp + Gly222AsnGly205Ser + Leu209Thr + Thr217Gln + Ile220Met +
Gly222Gln + Thr223GluGly203Asn + Pro204Ser + Gly205Pro + Trp212Pro +
Gly215Gln + Thr217GluPro204Ser + Gly205Ser + Leu209Asn + Ser216Glu +
Ile220Leu + Gly222AsnGly203Gln + Leu209Thr + Trp212Leu + Ile213Gly +
Ser219Glu + Ile220HisIle208Gln + Trp212Gly + Ile213Asn + Thr217Pro +
Gly222Ser + Thr223AspPro204Ser + Ile208His + Thr211Gly + Ile213Met +
Gly214Gln + Ser216AspGly203Asn + Gly205Pro + Leu209Glu + Trp212Ile +
Ile213Val + Gly215ProGly203Asn + Pro204Gln + Leu209Met + Trp212Ser +
Thr217Glu + Arg218GluLeu209Gln + Trp212Gln + Gly214Asn + Gly215Ser +
Gly222Glu + Thr223GluGly205Pro + Thr211Gly + Trp212Val + Arg218Glu +
Ser219Asp + Ile220ProGly203Asn + Pro204Gln + Gly205Gln + Gly214Pro +
Arg218Glu + Ser219AspGly205Pro + Thr211Gln + Gly215Asn + Thr217Ser +
Arg218Glu + Ser219GluPro204Asn + Gly205Ser + Ile208Gly + Thr217Ser +
Arg218Glu + Ser219AspThr206Pro + Gly214Ser + Thr217Gln + Arg218Glu +
Ser219Glu + Thr223AsnThr206Gln + Leu209Glu + Thr211Asn + Trp212Ile +
Gly215Ser + Ser219AspLeu209Glu + Thr211Gln + Ile213Val + Gly215Gln +
Ser219Glu + Gly222ProPro204Gln + Ser207Glu + Ile208Pro + Thr211Gly +
Ile220Pro + Ser221AspPro204Asn + Thr206Gln + Ser207Glu + Ile208Ala +
Leu209Cys + Ile220GluIhr206Glu + Thr211Gln + Ile213Pro + Gly214Asn +
Ser221Asp + Gly222AspGly203Asn + Pro204Gln + Leu209Asp + Gly215Pro +
Ser219Asp + Ile220AspSer207Glu + Ile208Ala + Thr211Gly + Trp212Ser +
Ser219Glu + Ile220GlnSer207Glu + Leu209Gly + Thr211Pro + Gly215Asn +
Ser219Glu + Ile220ValSer207Glu + Ile208Asn + Leu209Cys + Gly215Asn +
Ser219Glu + Thr223SerGly203Asn + Gly205Ser + Ser207Asp + Thr217Ser +
Ser219Asp + Ile220LeuGly203Asn + Pro204Asn + Ser207Asp + Leu209Thr +
Ser219Asp + Thr223GlnGly203Pro + Ile208Pro + Thr211Ser + Ser221Asp +
Gly222Asp + Thr223AspGly205Ser + Ser207Glu + Leu209Glu + Thr217Ser +
Ser219Glu + Gly222AsnThr206Pro + Leu209Ser + Thr211Pro + Ile213Glu +
Gly215Glu + Ile220AlaGly203Ser + Ile208Ser + Leu209His + Trp212Gly +
Gly214Asp + Ser216GluGly203Ser + Gly205Ser + Trp212Ala + Gly214Glu +
Gly215Ser + Ser216AspGly205Gln + Ser207Glu + Ile208Ser + Leu209Gly +
Ser219Asp + Ser221GluThr206Pro + Ser207Glu + Ile208His + Trp212Ala +
Ser219Glu + Ser221AspGly205Ser + Ser207Asp + Leu209Cys + Thr217Pro +
Ser221Asp + Gly222GluSer207Glu + Leu209Asp + Thr211Gln + Trp212Gln +
Thr217Gln + Ile220GluLeu209Asp + Thr211Gln + Gly214Pro + Thr217Glu +
Ser219Glu + Thr223GlnGly203Ser + Thr206Glu + Ser207Glu + Gly215Ser +
Ser219Asp + Ile220ThrGly205Ser + Thr206Glu + Ser207Glu + Thr211Gly +
Ile220Val + Gly222GluThr206Glu + Ser207Asp + Leu209Ser + Trp212Ile +
Ile220Thr + Gly222GluGly203Pro + Ser207Glu + Leu209Asn + Thr217Gln +
Arg218Asp + Ser219AspGly205Gln + Thr206Gln + Ser207Asp + Arg218Glu +
Ser219Asp + Ile220ProPro204Ser + Gly205Gln + Ser207Asp + Thr217Asn +
Arg218Asp + Ser219AspGly203Pro + Thr206Pro + Ser207Glu + Ile208Asn +
Leu209Asp + Thr217SerGly203Asn + Pro204Gln + Thr206Asp + Trp212Phe +
Ser221Glu + Thr223AspPro204Gln + Gly205Gln + Ser207Glu + Leu209Glu +
Trp212Met + Ser221GluGly203Asn + Trp212Phe + Gly214Ser + Ser219Asp +
Ser221Glu + Thr223GlnGly205Gln + Thr206Gln + Ile208Pro + Thr217Pro +
Ser219Asp + Ser221AspGly203Asn + Thr211Asn + Gly215Pro + Ser219Asp +
Ser221Asp + Gly222AsnPro204Ser + Trp212His + Gly214Gln + Ser219Glu +
Ser221Glu + Gly222GlnGly203Pro + Thr211Gln + Ile213Met + Ser219Glu +
Ser221Asp + Gly222SerIle208Leu + Thr211Gly + Gly214Pro + Gly215Asn +
Ser216Glu + Arg218AspIle208Ala + Leu209Pro + Trp212Pro + Ser216Glu +
Arg218Asp + Thr223ProGly205Gln + Ile208Asn + Gly215Ser + Ser216Glu +
Arg218Glu + Thr223AsnGly203Ser + Thr206Gly + Gly214Pro + Gly215Gln +
Ser216Asp + Arg218AspGly203Ser + Thr206Pro + Trp212Pro + Gly215Asn +
Ser216Glu + Arg218GluGly203Asn + Gly205Asn + Leu209Val + Ile220Glu +
Gly222Glu + Thr223SerPro204Gly + Gly205Pro + Leu209Ile + Ile213Met +
Ser221Asp + Thr223GluGly205Ser + Trp212Tyr + Gly214Pro + Arg218Glu +
Ser219Asp + Ser221AspGly205Gln + Ile208Ala + Leu209Cys + Arg218Glu +
Ser219Asp + Ser221AspGly205Gln + Leu209Val + Trp212Cys + Ile220Asp +
Ser221Glu + Thr223AspPro204Gln + Thr211Gln + Gly215Glu + Thr217Asp +
Arg218Glu + Thr223AsnGly203Gln + Leu209Asn + Ser216Asp + Arg218Glu +
Ser219Asp + Thr223ProIle208Ala + Leu209Pro + Gly214Ser + Ser216Asp +
Arg218Glu + Ser219AspPro204Asn + Ile208Gly + Thr211Gln + Ser216Asp +
Arg218Glu + Ser219GluGly203Ser + Ile208Ala + Trp212Ser + Ser216Glu +
Arg218Asp + Ser219GluGly205Pro + Thr211Ser + Ser216Glu + Thr217Asp +
Ser219Asp + Gly222AsnPro204Gln + Leu209Ala + Gly215Gln + Ser216Glu +
Thr217Asp + Ser219AspPro204Asn + Ser207Glu + Thr211Gln + Gly214Ser +
Gly222Glu + Thr223AsnGly205Asn + Ser207Asp + Trp212His + Gly214Ser +
Gly222Glu + Thr223GluGly203Ser + Ser207Glu + Thr211Ser + Ile213Ala +
Arg218Asp + Ser221AspGly203Gln + Thr206Glu + Trp212Ile + Ile213Asn +
Ser219Asp + Gly222GluIle208Ala + Leu209Met + Thr211Pro + Ile213Thr +
Gly214Glu + Thr217GluGly203Ser + Pro204Ser + Gly205Ser + Gly214Asp +
Gly215Pro + Thr217GluGly203Asn + Ile208Met + Ile213Cys + Gly214Asp +
Thr217Glu + Thr223AsnPro204Asn + Leu209Ile + Trp212Val + Ile213Asp +
Gly215Asp + Arg218GluSer207Asp + Ile208Met + Thr211Pro + Trp212Ala +
Gly215Asn + Arg218GluPro204Asn + Thr206Gln + Ser207Glu + Gly214Asn +
Gly215Gln + Arg218GluGly203Gln + Gly205Pro + Ser207Asp + Ile208Gly +
Arg218Glu + Gly222GlnGly205Pro + Thr206Asn + Ser207Asp + Trp212Tyr +
Arg218Asp + Ile220ThrPro204Asn + Gly205Pro + Ser207Glu + Ile208Gln +
Thr217Pro + Arg218AspThr206Gln + Ser207Glu + Trp212Ser + Thr217Asn +
Arg218Glu + Ile220AlaGly205Pro + Thr206Glu + Leu209Thr + Gly214Gln +
Arg218Asp + Ser219GluGly203Ser + Pro204Asn + ser207Asp + Gly215Gln +
Thr217Asp + Ile220AspIle208Gly + Thr211Gln + Ile213Glu + Arg218Glu +
Gly222Ser + Thr223GlnGly203Pro + Thr206Gly + Gly214Gln + Ser219Glu +
Ser221Asp + Thr223AspThr211Gly + Trp212Phe + Ser219Asp + Ile220Pro +
Ser221Glu + Thr223GluLeu209Ala + Ile213Ala + Ser219Asp + Ile220Gln +
Ser221Asp + Thr223AspPro204Gln + Leu209Glu + Trp212Met + Gly214Ser +
Gly215Asp + Thr217GluIle208Met + Ile213Ser + Thr217Glu + Ile220Asp +
Ser221Asp + Thr223GlnLeu209Asp + Thr211Asn + Ile213Asp + Arg218Glu +
Ile220Leu + Gly222SerGly203Pro + Gly205Gln + Thr217Asp + Arg218Glu +
Ile220Ala + Ser221AspTrp212Cys + Ile213Gly + Arg218Glu + Ser219Glu +
Ile220Pro + Gly222GluThr206Gln + Ile208Gly + Leu209Ile + Arg218Glu +
Ser219Glu + Gly222AspGly203Gln + Ile208Met + Ser219Glu + Ile220Glu +
Gly222Pro + Thr223GluGly203Gln + Gly205Asn + Thr211Gln + Ser219Asp +
Ile220Glu + Thr223GluPro204Asn + Gly205Pro + Leu209Ser + Gly214Ser +
Arg218Glu + Ser221GluIle208Thr + Leu209Asn + Trp212Asn + Arg218Asp +
Ser221Glu + Gly222AsnGly203Ser + Pro204Gln + Gly205Asn + Thr211Gly +
Arg218Asp + Ser221GluPro204Asn + Thr206Pro + Gly214Pro + Arg218Glu +
Ser221Glu + Thr223AsnThr206Asp + Ile213Gln + Gly215Asn + Thr217Asp +
Ser219Asp + Thr223SerIle213Met + Gly214Asp + Gly215Ser + Thr217Glu +
Ser219Asp + Ile220SerPro204Asn + Gly205Gln + Ser207Asp + Gly215Pro +
Ser219Glu + Thr223GluPro204Ser + Thr206Gln + Ser207Glu + Thr217Gly +
Ser219Asp + Thr223AspPro204Gln + Trp212Leu + Gly215Glu + Thr217Pro +
Arg218Asp + Ser219AspGly203Asn + Thr211Gly + Gly214Asn + Gly215Pro +
Ser219Asp + Gly222AspGly203Pro + Trp212Ile + Gly214Asn + Ser219Glu +
Ile220Pro + Gly222GluThr206Glu + Leu209Ile + Gly215Asn + Ser219Asp +
Ile220Cys + Thr223AspGly203Asn + Ser207Asp + Gly214Gln + Ser216Glu +
Arg218Asp + Ile220ThrSer207Glu + Ile208Cys + Ile213His + Gly214Ser +
Ser216Asp + Arg218GluThr206Gln + Ile208Ser + Leu209Asp + Gly215Glu +
Arg218Asp + Ile220AlaGly205Ser + Ile213Cys + Gly214Asp + Arg218Glu +
Ser219Asp + Gly222GlnPro204Asn + Gly214Asp + Arg218Asp + Ser219Asp +
Gly222Pro + Thr223GlnPro204Gly + Thr211Asn + Ile213Ala + Gly215Asp +
Arg218Glu + Ile220GlySer207Glu + Leu209Ile + Ser216Glu + Thr217Gly +
Ser219Asp + Ile220GlyGly205Ser + Ser207Asp + Thr211Gly + Ser216Glu +
Ser219Asp + Ile220AsnPro204Gln + Gly205Ser + Thr206Asn + Leu209Glu +
Trp212Gly + Ser216AspGly205Pro + Trp212Gly + Ile213His + Gly214Asp +
Ser216Glu + Ser219GluPro204Ser + Thr206Asn + Ile213Ala + Ser216Asp +
Ser219Glu + Thr223GlnIle208Asn + Trp212Asn + Gly215Asn + Ser216Glu +
Ser219Glu + Ile220SerIle208Leu + Thr211Gly + Gly214Gln + Ser216Glu +
Ser219Glu + Gly222ProPro204Ser + Thr206Ser + Ser216Asp + Thr217Pro +
Ser219Glu + Thr223AsnLeu209Val + Trp212Cys + Gly214Ser + Arg218Glu +
Ile220Glu + Thr223AspGly205Gln + Trp212Met + Gly215Asp + Arg218Glu +
Ile220Glu + Gly222SerGly205Pro + Thr206Ser + Ser207Glu + Leu209Gly +
Gly214Ser + Thr223GluThr206Asp + Leu209Glu + Thr211Gln + Thr217Gln +
Ile220Met + Thr223GluPro204Gly + Ile208Cys + Thr217Ser + Arg218Glu +
Ser221Glu + Thr223AspSer207Glu + Leu209Val + Ile213Gly + Ser216Glu +
Thr217Asp + Gly222GlnIle208Thr + Ser216Glu + Ser219Glu + Ile220Leu +
Ser221Glu + Thr223ProGly203Gln + Gly205Gln + Ile213Ala + Ser216Glu +
Ser219Asp + Ser221AspGly203Gln + Pro204Gln + Thr206Asp + Leu209Asn +
Arg218Glu + Gly222GluGly205Gln + Ile213Ala + Gly215Glu + Ser216Glu +
Ile220Glu + Gly222AsnThr206Asn + Ile208Ala + Leu209Asp + Ile213Glu +
Gly214Glu + Gly222SerGly203Ser + Pro204Ser + Ile208Cys + Ile213Glu +
Gly215Glu + Ser219GluThr206Pro + Leu209Cys + Ile213Glu + Gly215Glu +
Thr217Ser + Ser219Glu
表28
环区6-七替换突变体Pro204Asn + Leu209Thr + Thr211Gln + Trp212His+
Gly215Asp + Ile220Val + Thr223SerPro204Gly + Gly205Asn + Ile208Leu + Thr211Asn +
Trp212Gly + Gly214Pro + Arg218AspGly203Pro + Thr206Gly + Ile208Pro + Thr211Gln +
Thr217Glu + Ile220Met + Thr223AsnPro204Gln + Ile208Ala + Leu209Gln + Thr211Ser +
Gly214Ser + Ser219Glu + Thr223ProPro204Gly + Gly205Pro + Thr206Glu + Ile208Pro +
Leu209Val + Ile213His + Ile220LeuGly205Gln + Thr206Ser + Ile208Leu + Thr211Ser +
Gly214Asn + Gly222Pro + Thr223GluPro204Gly + Gly205Ser + Ile208Ala + Trp212Leu +
Thr217Gln + Arg218Asp + Thr223AsnIle208Asn + Thr211Asn + Trp212Val + Ile213Asp +
Ile220Ala + Gly222Pro + Thr223ProPro204Gly + Ile208Cys + Trp212Cys + Ile213Gly +
Thr217Gln + Ser219Asp + Ile220AlaGly205Pro + Ile208Asn + Thr211Pro + Trp212Phe +
Ile220Met + Ser221Glu + Gly222SerGly203Gln + Pro204Ser + Thr206Ser + Ile208Val +
Trp212Tyr + Ile220Gly + Ser221GluPro204Ser + Gly205Pro + Leu209Met + Trp212Leu +
Ile213Asp + Gly215Gln + Gly222SerGly205Pro + Ile208Pro + Leu209Thr + Thr211Ser +
Trp212Asn + Ser216Glu + Ile220AlaGly203Gln + Gly205Ser + Thr206Gly + Trp212Ala +
Gly215Pro + Thr217Ser + Ser221GluGly203Ser + Ile208Cys + Leu209Met + Trp212Ser +
Gly215Pro + Ser216Glu + Thr223ProGly205Ser + Thr206Gly + Trp212Gly + Thr217Gly +
Arg218Glu + Ile220Gly + Gly222AsnGly203Ser + Gly205Pro + Thr206Gly + Gly214Gln +
Ser216Asp + Ile220Gly + Thr223SerPro204Asn + Trp212Gln + Ile213Thr + Gly215Ser +
Ser216Glu + Thr217Asn + Ile220AsnGly203Asn + Pro204Gly + Thr206Asn + Ile208Leu +
Thr211Pro + Ile220Leu + Gly222ProGly203Asn + Gly205Asn + Thr211Ser + Trp212Met +
Ile213Glu + Gly214Asn + Thr223AsnPro204Asn + Gly205Pro + Ile208Ser + Thr211Gln +
Ile213Gln + Gly215Gln + Arg218GluGly203Gln + Gly205Gln + Thr206Ser + Ile208Val +
Trp212Gln + Thr217Pro + Ile220GluPro204Gly + Gly205Pro + Ile208Met + Thr211Ser +
Ile213Gly + Gly215Pro + Ser216GluGly203Gln + Gly205Ser + Thr206Ser + Ile208Ala +
Ile213Ala + Ile220Met + Thr223GluPro204Gly + Thr206Gln + Leu209Ala + Thr211Gln +
Thr217Asn + Arg218Glu + Ile220ProGly203Gln + Pro204Gly + Gly205Ser + Ser207Asp +
Leu209Asn + Gly214Ser + Gly215ProGly203Gln + Gly205Gln + Leu209Met + Trp212Thr +
Gly214Gln + Ile220Asn + Gly222GluGly203Ser + Thr206Asp + Ile208Leu + Thr211Pro +
Gly214Ser + Gly215Ser + Gly222GlnPro204Gln + Thr206Gly + Ile208Asn + Trp212Gly +
Ile213Pro + Thr217Pro + Arg218AspGly205Asn + Ile208Thr + Thr211Gly + Ile213His +
Thr217Gly + Arg218Glu + Thr223AsnGly205Asn + Thr206Pro + Ile208Ala + Leu209Ala +
Trp212Leu + Gly215Pro + Gly222AspGly203Pro + Thr206Gln + Ile208Asn + Leu209Cys +
Gly215Glu + Gly222Pro + Thr223SerPro204Gln + Ile208Ala + Trp212Asn + Ile213Ser +
Gly215Ser + Ser219Asp + Ile220AspGly203Ser + Thr211Ser + Trp212Met + Arg218Asp +
Ser219Asp + Gly222Asn + Thr223AsnGly203Ser + Gly205Pro + Thr206Pro + Ile208Leu +
Arg218Glu + Ser219Glu + Ile220AlaPro204Gly + Thr211Asn + Gly214Pro + Ser216Asp +
Thr217Glu + Ile220Gln + Gly222ProGly203Asn + Gly205Pro + Leu209Val + Thr211Pro +
Gly215Asn + Ser216Glu + Thr217AspPro204Gly + Gly205Gln + Thr211Ser + Trp212Met +
Gly215Asp + Ser216Glu + Thr217ProPro204Asn + Thr206Gly + Ile208Met + Leu209Asp +
Ser219Glu + Ile220His + Thr223ProGly205Gln + Thr206Glu + Ile208Thr + Leu209Thr +
Thr211Gln + Trp212Cys + Ser221AspPro204Gly + Thr206Glu + Leu209Ala + Trp212Met +
Ile220Val + Ser221Asp + Gly222SerGly205Gln + Thr206Glu + Thr211Asn + Trp212Ser +
Thr217Gln + Ser221Asp + Thr223GlnSer207Glu + Leu209Thr + Ile213Gln + Gly214Gln +
Gly215Asn + Ser219Glu + Ile220GluThr206Gln + Ser207Glu + Leu209Ile + Ile213Ala +
Gly214Ser + Ile220Thr + Ser221AspPro204Asn + Thr206Gln + Ser207Glu + Ile208Ala +
Leu209Cys + Thr211Gly + Ile220GluThr206Pro + Ser207Glu + Thr211Gln + Gly214Pro +
Gly215Ser + Thr217Gly + Ser219GluPro204Asn + Ser207Glu + Leu209Gly + Thr211Asn +
Gly215Asn + Ser219Glu + Ile220ValPro204Gln + Gly205Ser + Leu209Ser + Trp212Gly +
Thr217Glu + Arg218Glu + Ser219AspGly203Gln + Ser207Asp + Leu209His + Gly214Ser +
Gly215Gln + Ser219Asp + Ser221AspPro204Gln + Gly205Asn + Ser207Glu + Leu209Gln +
Thr211Gly + Ser219Glu + Ser221AspGly203Asn + Thr206Glu + Ile208Gln + Leu209Gln +
Thr211Pro + Trp212Ile + Gly222GluGly203Ser + Thr206Glu + Ser207Asp + Thr211Ser +
Trp212Gly + Ile220Gly + Gly222AspGly205Pro + Thr206Asp + Ser207Asp + Ile213Pro +
Gly214Ser + Gly222Glu + Thr223AsnGly203Gln + Thr206Asp + Leu209Met + Trp212Leu +
Gly214Asn + Gly222Asp + Thr223GluPro204Asn + Thr206Gln + Leu209Ser + Ser216Glu +
Thr217Asp + Arg218Glu + Ser219AspPro204Asn + Thr206Gly + Ser207Glu + Ile208Thr +
Gly215Gln + Ile220Asp + Gly222GluGly203Pro + Gly205Gln + Thr206Ser + Leu209Asp +
Thr211Ser + Ile213Cys + Thr217AspGly205Gln + Ser207Glu + Thr211Ser + Trp212Ser +
Ile213Gln + Arg218Asp + Ile220GluGly203Ser + Pro204Gly + Thr206Glu + Ser207Glu +
Ile208Ser + Leu209Asp + Trp212AlaGly203Ser + Ile208Thr + Leu209Glu + Trp212Tyr +
Thr217Gln + Ser219Asp + Ser221GluGly203Ser + Gly205Ser + Thr211Ser + Gly215Gln +
Thr217Pro + Ser219Glu + Ser221AspGly205Gln + Thr206Gly + Leu209Ser + Ile213Gly +
Ser219Glu + Ser221Asp + Thr223GlnPro204Gln + Ser207Asp + Ile208Ser + Ile213Leu +
Arg218Glu + Ser219Glu + Ser221GluGly203Asn + Trp212Gln + Gly214Pro + Ser216Glu +
Arg218Glu + Ile220Leu + Thr223ProPro204Asn + Gly205Gln + Thr206Gly + Ile213His +
Gly214Ser + Ser216Asp + Arg218GluIle208Val + Ile213Met + Gly214Ser + Ser216Asp +
Thr217Gly + Arg218Asp + Ile220MetPro204Ser + Gly205Asn + Ile208Leu + Leu209His +
Ser216Asp + Arg218Glu + Thr223ProGly203Gln + Ile208Cys + Thr211Pro + Trp212Ser +
Gly215Ser + Ser216Glu + Arg218GluGly203Pro + Gly205Ser + Leu209Ile + Thr211Asn +
Thr217Glu + Ser219Glu + Ile220HisGly205Ser + Leu209Cys + Thr211Asn + Gly215Gln +
Thr217Glu + Ser219Asp + Gly222ProGly203Gln + Ile208Ala + Trp212Ile + Gly215Gln +
Thr217Glu + Ser219Asp + Gly222ProGly203Ser + Thr206Glu + Ser207Glu + Ile208Pro +
Ile213Thr + Gly222Glu + Thr223AspGly203Ser + Ile208Gln + Trp212Ser + Ile213Leu +
Arg218Asp + Ser219Glu + Ser221AspGly203Gln + Gly205Asn + Trp212Asn + Ile213Glu +
Ser216Glu + Thr217Pro + Arg218AspThr206Asn + Leu209Asp + Ile213Met + Ser216Glu +
Thr217Glu + Ser219Glu + Ile220GlyGly205Gln + Thr211Pro + Trp212Val + Gly214Ser +
Ser216Asp + Arg218Glu + Ser219GluThr206Asn + Ser207Asp + Thr211Ser + Ile213Cys +
Ile220Met + Ser221Asp + Thr223AspGly203Asn + Ile213Ser + Thr217Asp + Arg218Glu +
Ser219Asp + Ser221Asp + Thr223GlnThr206Asn + Ser207Glu + Leu20gCys + Trp212Ser +
Arg218Glu + Ser221Asp + Gly222ProGly203Gln + Ser207Asp + Gly214Pro + Gly215Pro +
Arg218Asp + Ser221Asp + Gly222SerGly203Asn + Thr206Glu + Leu209Gln + Arg218Glu +
Ser219Asp + Ser221Glu + Gly222ProGly203Pro + Ser207Glu + Trp212Ser + Gly214Pro +
Ser219Glu + Ser221Asp + Thr223AspGly205Gln + Thr211Gln + Ile213Glu + Ser216Glu +
Arg218Asp + Ser219Asp + Ile220AlaGly203Asn + Pro204Ser + Ser207Glu + Gly215Gln +
Thr217Asp + Ile220Asp + Ser221AspGly203Gln + Gly205Asn + Ser207Asp + Leu209Ile +
Thr211Gly + Thr217Asp + Arg218GluPro204Gly + Trp212Met + Gly214Asp + Ser216Asp +
Thr217Glu + Ser219Glu + Thr223GlnSer207Asp + Trp212Tyr + Gly215Asn + Ser216Asp +
Arg218Glu + Ser219Glu + Thr223ProThr206Gly + Ser207Asp + Ile208Gln + Ser216Glu +
Arg218Glu + Ser219Asp + Ile220GlnSer207Glu + Leu209Gln + Thr211Pro + Gly215Pro +
Ser216Asp + Arg218Glu + Ser219AspGly205Asn + Ser207Asp + Ile208Val + Gly214Pro +
Ser216Asp + Arg218Glu + Ser219GluThr206Asp + Ile208His + Thr211Ser + Trp212Asn +
Arg218Asp + Ser219Glu + Gly222ProIle208Gly + Trp212Met + Ile213Gln + Ser216Glu +
Arg218Asp + Ser219Asp + Ser221AspGly203Asn + Thr206Glu + Leu209Asp + Thr217Pro +
Ile220Ser + Gly222Glu + Thr223AspGly203Asn + Thr206Asp + Ile208His + Thr211Ser +
Trp212Phe + Arg218Asp + Ser221GluPro204Asn + Gly214Asn + Gly215Pro + Ser216Glu +
Thr217Asp + Arg218Asp + Ser221AspPro204Gln + Gly205Pro + Thr211Asn + Gly215Gln +
Ser219Glu + Ser221Asp + Thr223GluSer207Asp + Leu209Val + Trp212Cys + Gly214Ser +
Arg218Glu + Ile220Glu + Thr223AspThr206Asn + Ser207Asp + Ile208Ala + Gly215Pro +
Arg218Glu + Ser221Glu + Thr223AspPro204Gln + Gly205Asn + Thr206Gln + Leu209Cys +
Thr217Asp + Arg218Glu + Ser221GluGly203Pro + Thr206Ser + Ile213His + Ser219Glu +
Ile220Cys + Gly222Glu + Thr223AspGly205Ser + Thr211Ser + Trp212Gln + Gly214Ser +
Ser219Asp + Ile220Glu + Thr223AspSer207Asp + Leu209Gln + Ile213Gly + Ser216Glu +
Ser219Asp + Ile220Ser + Ser221GluSer207Asp + Thr211Pro + Trp212Gln + Ile213Pro +
Ser216Asp + Ser219Asp + Ser221AspPro204Gly + Gly205Pro + Ile208Gly + Leu209Glu +
Ile213Thr + Thr217Asp + Ser221GluThr206Gln + Ser207Asp + Ile208Cys + Leu209Thr +
Arg218Asp + Ser219Asp + Thr223AspGly203Pro + Ile208Asn + Thr211Gln + Ile213Met +
Thr217Gln + Arg218Glu + Ser221AspGly203Asn + Gly205Asn + Thr206Gln + Thr211Gly +
Trp212Gln + Arg218Glu + Ser221GluGly203Ser + Ile208Pro + Gly215Pro + Arg218Asp +
Ile220Asn + Ser221Glu + Thr223AsnThr206Pro + Thr211Pro + Gly214Ser + Gly215Pro +
Arg218Asp + Ser221Glu + Thr223SerThr206Asp + Ser207Asp + Trp212Thr + Ile213Cys +
Ser216Glu + Thr217Pro + Ser219AspGly205Asn + Thr206Asp + Ser207Asp + Ile213Thr +
Ser216Glu + Ser219Glu + Ile220LeuThr206Ser + Ile213Met + Gly214Asp + Gly215Ser +
Thr217Glu + Ser219Asp + Ile220GluPro204Asn + Thr206Pro + Leu209Glu + Thr217Gly +
Arg218Glu + Ser219Asp + Thr223AspPro204Gly + Ser207Glu + Thr211Pro + Gly214Asp +
Arg218Asp + Ser219Asp + Thr223GlnGly205Pro + Leu209Glu + Thr211Asn + Gly214Asp +
Thr217Asp + Ile220Met + Gly222AsnPro204Ser + Trp212His + Ile213Asn + Gly214Glu +
Thr217Glu + Ser219Glu + Gly222AsnGly203Gln + Ser207Glu + Ile208Cys + Gly215Pro +
Ser219Glu + Ile220Asn + Thr223GluSer207Glu + Ile208Gly + Ile213Pro + Arg218Asp +
Ile220Met + Gly222Asp + Thr223GluThr206Asp + Ser207Glu + Ile208Gly + Thr211Gln +
Ser216Asp + Ser221Glu + Thr223AsnGly203Asn + Ser207Glu + Trp212Ser + Ile213Asp +
Gly215Asn + Ser216Glu + Arg218GluGly203Asn + Leu209Ser + Ile213Val + Thr217Pro +
Ser219Asp + Ile220Ala + Gly222GluIle208Met + Thr211Gly + Trp212Leu + Gly214Pro +
Gly215Pro + Ser219Asp + Gly222GluSer207Glu + Leu209Ser + Ile213Ser + Gly214Asn +
Ser216Asp + Arg218Glu + Ser221AspGly203Ser + Ser207Asp + Ile208Asn + Thr211Asn +
Gly214Glu + Ser219Asp + Ile220AspSer207Asp + Thr211Asn + Ile213Ala + Gly215Asp +
Thr217Ser + Ser219Glu + Ile220GluPro204Ser + Gly205Asn + Thr211Pro + Thr217Glu +
Ser221Asp + Gly222Glu + Thr223GluSer207Glu + Leu209Ala + Gly214Pro + Ser216Glu +
Thr217Ser + Arg218Glu + Ile220LeuIle208Val + Leu209Glu + Trp212Ser + Gly215Glu +
Ser219Asp + Ile220Asp + Gly222ProGly203Gln + Ile213Ser + Gly215Glu + Ser216Asp +
Ser219Asp + Gly222Ser + Thr223GlnGly205Pro + Leu209Val + Gly214Asn + Ser216Glu +
Ser219Asp + Ile220Glu + Gly222AspPro204Asn + Leu209Asp + Ile213Met + Gly214Pro +
Thr217Glu + Ser221Glu + Gly222AspGly203Ser + Ser207Glu + Trp212Pro + Gly214Asp +
Ser216Asp + Thr217Asp + Ile220MetThr206Glu + Thr211Gln + Ile213Pro + Gly214Asp +
Thr217Gly + Ser221Asp + Gly222AspIle208Cys + Leu209Ser + Thr211Asn + Ile213Ala +
Gly215Asp + Arg218Glu + Ile220GlyIle208Thr + Leu209His + Ile213Met + Gly214Glu +
Ser221Glu + Gly222Asp + Thr223AspThr206Gln + Gly214Gln + Gly215Glu + Thr217Gly +
Arg218Glu + Ser219Glu + Ser221AspPro204Ser + Thr206Ser + Ile213Cys + Gly215Glu +
Ser216Glu + Thr217Glu + Thr223AspThr206Gln + Ile208Met + Trp212Gly + Gly214Asp +
Ser216Asp + Thr217Pro + Ser219AspSer207Glu + Ile208Thr + Thr211Gln + Trp212Gln +
Gly215Asp + Ser219Asp + Ser221AspGly203Ser + Pro204Gln + Ser207Glu + Ile208Cys +
Gly214Glu + Ser219Asp + Ser221GluSer207Asp + Ile208Thr + Thr211Asn + Gly215Glu +
Ser219Glu + Ile220Met + Ser221GluPro204Ser + Ser207Glu + Gly214Ser + Gly215Asp +
Ser219Asp + Ile220Met + Ser221GluSer207Asp + Ile208Leu + Trp212Ile + Ile213Asp +
Thr217Asn + Arg218Glu + Ser221GluThr206Glu + Trp212Ser + Ile213Cys + Thr217Gly +
Arg218Glu + Ser219Glu + Thr223GluThr206Asp + Ser207Glu + Trp212Cys + Ile213Cys +
Gly215Asn + Ser216Asp + Arg218GluThr206Glu + Ser207Glu + Gly214Gln + Gly215Pro +
Ser216Asp + Thr217Ser + Arg218AspPro204Ser + Gly205Ser + Trp212Pro + Ser216Asp +
Ser219Asp + Ile220Val + Thr223SerGly203Gln + Leu209Gln + Trp212Ala + Gly215Asn +
Arg2l8Glu + Ile220Glu + Thr223GluGly203Gln + Leu209Asp + Thr211Asn + Gly214Glu +
Arg218Glu + Ile220Leu + Gly222SerGly203Ser + Pro204Gln + Thr211Pro + Gly215Asp +
Arg218Glu + Ile220Glu + Thr223AsnThr206Asn + Ile208Gln + Ser216Glu + Thr217Gly +
Arg218Asp + Ile220Gly + Ser221AspPro204Ser + Ile208His + Gly214Pro + Ser216Glu +
Arg218Glu + Ser221Glu + Thr223SerPro204Gln + Leu209Ile + Trp212Ala + Ser216Asp +
Thr217Ser + Arg218Asp + Ser221AspGly203Pro + Pro204Asn + Ser207Asp + Ile208Cys +
Thr217Gly + Ile220Gly + Thr223GluPro204Ser + Ser207Asp + Leu209His + Thr211Ser +
Gly215Asn + Gly222Ser + Thr223GluIle208His + Thr211Pro + Trp212Leu + Thr217Ser +
Arg218Asp + Ser221Asp + Thr223GluGly205Asn + Thr206Asn + Ser207Asp + Ile213Thr +
Ser216Asp + Ser219Asp + Gly222GluThr206Asp + Ser207Glu + Ile208Leu + Trp212Gly +
Ser216Glu + Thr217Gln + Gly222AspGly205Asn + Thr206Asn + Ser207Asp + Ile208Gln +
Trp212Cys + Ser216Glu + Thr217GluSer207Glu + Ile208Asn + Trp212Thr + Ile213Gln +
Ser216Asp + Thr217Glu + Thr223GlySer207Asp + Ile208His + Thr211Asn + Gly215Asn +
Ser216Asp + Thr217Glu + Thr223GlyGly203Ser + Pro204Ser + Ile213Thr + Ser216Glu +
Thr217Gly + Ser219Asp + Ser221GluGly203Asn + Ile208Ser + Thr211Asn + Ser216Asp +
Ser219Glu + Ser221Asp + Thr223GlyGly203Ser + Thr206Pro + Thr211Gly + Trp212Thr +
Ser216Glu + Ser219Glu + Ser221AspTrp212Ser + Gly214Pro + Gly215Asp + Thr217Asp +
Ser219Glu + Ser221Asp + Thr223ProThr206Ser + Ser207Asp + Ile208Pro + Leu209Glu +
Gly215Asp + Ile220Thr + Ser221GluGly203Asn + Trp212Tyr + Gly214Asp + Gly215Ser +
Arg218Glu + Ile220Pro + Thr223SerPro204Ser + Ile208Thr + Thr211Gly + Trp212His +
Gly214Asp + Thr217Pro + Arg218GluLeu209Glu + Thr21lPro + Ile213Cys + Gly214Glu +
Arg218Asp + Ile220Asn + Ser221AspGly205Gln + Thr211Asn + Gly214Asp + Gly215Glu +
Thr217Asp + Ile220Ala + Thr223GluGly205Gln + Thr211Gln + Ile213Asn + Gly215Asp +
Thr217Glu + Ile220Glu + Ser221AspPro204Asn + Ile213Asp + Thr217Glu + Arg218Glu +
Ile220Ser + Gly222Pro + Thr223GluGly205Gln + Ser207Glu + Ile208Cys + Gly214Asp +
Ser216Glu + Ser219Asp + Thr223GlyGly203Ser + Ser207Glu + Gly215Asn + Ser216Asp +
Thr217Gly + Arg218Glu + Gly222GluThr206Gln + Leu209Met + Gly214Glu + Gly215Asn +
Ser219Glu + Ser221Asp + Gly222GluLeu209Met + Thr211Ser + Trp212Cys + Ser216Glu +
Thr217Pro + Ile220Glu + Ser221GluThr206Gly + Thr211Gln + Trp212Cys + Ser216Asp +
Ile220Glu + Ser221Asp + Thr223GlnGly203Ser + Pro204Asn + Ser207Glu + Ile213Asp +
Thr217Ser + Ser219Glu + Gly222AsnGly205Pro + Thr206Asp + Trp212Leu + Gly214Asn +
Ser216Glu + Ser219Asp + Gly222SerGly203Asn + Pro204Ser + Gly205Gln + Ser207Asp +
Trp212Phe + Gly215Asp + Arg218Glu
表29多环区双突变变异体Leu 96Gly + Leu209ProTyr104Cys + Ile220GlnLeu133Ile + Ser216GluLeu 96Ala + Asn162GluGln103Asp + Ile220ThrTyr169Ser + Ser190AspVa1 95Asp + Gly205GlnAsp 65Glu + Ile213AsnGly100Ser + Ile220GluGly100Glu + Asn163GlnAsn162Ser + Ser207AspSer170Glu + Ile220SerSer140Glu + Ile220LeuGln103Glu + Thr106ProLeu133Pro + Leu209AsnGly203Asn + Gly222AspSer207Glu + Ile213LeuSer101Asp + Thr206GlyGly203Ser + Ser207GluAsn 99Glu + Trp212AsnGly102Pro + Ser216AspSer219Asp + Ile220MetGly 70Asp + Leu133SerGly136Asn + Ser216AspArg167Glu + Leu209ProAsn194Glu + Leu209GlyLeu209Gln + Ser216GluGly 68Asn + Ser221GluAsp 65Glu + Ilel07CysArg218G1u + Ile220AlaAsp 65Glu + Gly135ProGly 68Asp + Phe192HisGly 66Asn + Leu209SerAsn168Ser + Gly214ProThr 71Gly + Leu 96GlyGly 70Gln + Ile220GlyTyr169His + Leu209IleThr217Pro + Ser219AspGly 70Pro + Ile220AspLeu 96Gln + Asn 99GluAsp 65Glu + Gly136SerAsn 99Glu + Ile213MetGly205Pro + Ser207AspAsn 99Glu + Leu209GlnVal 95Gln + Ser207AspVal 95Gln + Ser219AspAsn 67Ser + Thr217GluGly160Gln + Leu209AspGly136Asn + Gly160AsnAsn163Gln + Thr223AsnLeu209Glu + Thr217AsnGly102Pro + Trp212GlnArg 64Asp + Gly203AsnAsn 67Gln + Thr211AsnAsp 65Glu + Tyr137ProGln103Asp + Leu133IleLeu 96Glu + Ile213GlnAsn161Gln + Arg218GluLeu209Asn + Gly214ProGly 68Ser + Thr206AsnArg167Asp + Phe192IleAsp 65Glu + Thr211SerSer207Asp + Thr223GlySer207Asp + Gly222SerTyr104Leu + Arg167GluAsp 98Glu + Ile220AsnArg 64Glu + Gly215AsnAsn 67Asp + Asn163SerGly160Ser + Thr206GlnIle213Ser + Thr223AspGly215Gln + Ser219AspPhe202Leu + Ser219GluSer207Asp + Ile220CysTyr137Ser + Ser219AspGly100Asn + Leu209ThrTyr137His + Ser170AspSer170Asp + Ile220ThrThr106Gln + Ser207AspArg167Asp + Leu209ProSer105Asp + Gly136AsnSer139Asp + Phe192ThrSer190Glu + Pro204SerAsn 67Glu + Leu209AlaTrp212Ser + Ser216GluTyr104Ala + Thr206AspAsn161Gln + Gly222AsnVal 95Thr + Leu133SerGly160Ser + Asp165GluAsn163Ser + Leu209ValGly136Glu + Ile220ProSer191Asp + Pro204SerThr217Glu + Ile220AsnLeu209Met + Gly214AspThr 71Pro + Ser191GluSer101Glu + Trp212PheGly 70Ser + Ser219GluGly 66Gln + Asn 99SerAsn163Gln + Ser190GluIle107Ser + Ser190AspIle208Thr + Ser219AspSer216Asp + Ile220LeuSer140Asp + Ala164HisAsp 65Glu + Val 95SerPhe202Met + Leu209SerAsn 67Gln + Tyr169GlyGly136Ser + Asn162AspGly160Pro + Ser219AspGly102Pro + Ser207AspVal 95Asn + Ser207AspAsp 97Glu + Ile107GlnThr206Glu + Ile220ProSer216Glu + Thr223GlyAsp 97Glu + Leu133CysGly134Gln + Gly135AspThr206Gln + Ile220HisGly100Asn + Thr223AspGly 66Pro + Ile220ValGly 66Asn + Asn168GluSer190Asp + Ile213ValGly135Pro + Asn162GluLeu133Ile + Gly134ProAsn163Ser + Arg167GluGly134Gln + Ser216GluAsn194Gln + Leu209GlyAsn 99Gln + Ile213MetLeu 96Ile + Gly160AspArg167Glu + Ile220AlaVal 95Ala + Ser139AspGln103Ser + Gly214SerAsn 67Glu + Thr 71GlnGly135Pro + Ser191GluIle107Ala + Ile220GluGly214Gln + Ser219GluGly160Asp + Phe192MetLeu 96Ala + Asp 97GluAsn168Gln + Leu209GluGln103Glu + Gly136AsnGly215Asp + Gly222SerSer207Asp + Thr223AsnSer216Asp + Ile220CysVal 95Ser + Thr206GluGly 70Asn + Ser216AspLeu209Ser + Ile220AspAla164Pro + Ile208ThrTyr137Thr + Leu209GluThr106Asp + Tyr137ThrThr106Gly + Asp165GluAsn 99Asp + Ile213GlyAsn161Glu + Ile220SerGly160Asp + Ile220MetGly135Asp + Asn163GlnTyr169Ser + Ser216GluSer140Asp + Leu209AsnSer140Asp + Leu209GlnSer170Glu + Gly222ProIle213His + Ser216AspLeu 96Thr + Gly222AsnAsp 65Glu + Asn168GlnTyr104Leu + Ser219GluThr106Pro + Gly134ProAsn 99Asp + Gly215AsnThr206Glu + Ile220ValGly 70Pro + Leu 96GluThrl06G1y + Ser219GluGly100Glu + Ile107GlyGly102Gln + Tyr169GlyArg 64Glu + Pro204GlyLeu133Gln + Ser207AspGly100Gln + Leu133HisThr106Asp + Leu209AlaGly 68Glu + Ile220GlnAsn 67Asp + Gly 68GlnLeu 96Glu + Ile220LeuThr206Gln + Ser219AspAla164Gln + Gly222GlnGly 66Glu + Gly 68ProSer140Asp + Ala166AsnAsn 67Ser + Gly100AspSer219Asp + Ile220ThrSer170Glu + Gly215SerThr 71Gly + Phe192IleGly100Ser + Ser219AspGly215Pro + Ser219AspLeu209Met + Ser221AspAsn162Asp + Tyr169MetTyr104His + Asn162GluAsn 67Gln + Leu 96SerLeu209Asp + Ile220AlaGly 70Ser + Ile220GlnGly 66Ser + Gly 70GluTyr169His + Thr206GlyAsn194Gln + Thr206GlyIle107Glu + Leu209AsnGly 66Ser + Gly222GluLeu133Ile + Ser190GluSer105Asp + Ile220HisGly102Asp + Asn194GlnLeu 96Met + Ser216AspAsn161Asp + Gly203GlnGly136Gln + Asn161AspPhe202Ile + Ser219AspGln103Asp + Ile107ThrGly100Gln + Ser216AspPhe192Ala + Ser207GluGly136Pro + Ser138AspAsp 98Glu + Leu209ThrAsn194Ser + Ser216GluVal 95Asp + Gly222GlnThr211Pro + Ile220MetArg167Glu + Tyr169AlaGly 66Asn + Ser138AspAsn 67Glu + Leu209ProGly 70Glu + Tyr169ProGly102Pro + Leu209AsnGly 68Ser + Ser207AspSer101Glu + Ile220SerTyr137Val + Ser191GluThr211Gly + Arg218AspAsn161Gln + Asn194GlnThrl06Asn + Leu209GlnVal 95Ser + Gly134AspSer191Asp + Ile220SerAsn163Ser + Thr223GluLeu209Thr + Arg218AspGly 70Asp + Val 95GlyLeu209Met + Ser216AspThr206Ser + Arg218GluLeu209Ile + Thr217AspIle213Pro + Ser219GluGly205Ser + Thr206AspPhe192Gln + Thr206GluGly 68Asp + Leu209SerGly160Gln + Thr206GlyGly214Asp + Ile220LeuGly 70Asn + Arg218AspGly 70Glu + Gly136AsnLeu133Glu + Gly203ProGly135Gln + Ser207GluIle107Gly + Ile213GlnSer140Asp + Ile220LeuSer191Asp + Gly214GlnLeu209Ser + Gly214AspAsn 67Glu + Ile220GlnAla166Pro + Ser219AspGly102Ser + Ile220ValGln103Ser + Ser140GluGly102Asp + Ile220GlyTyr104Ile + Gly134GlnSer101Glu + Ile220CysAsn162Gln + Ser207AspVal 95Pro + Ser207AspAsn168Gln + Leu209AlaGly100Gln + Gly136AsnPhe192Pro + Asn194GluAsn 99Ser + Thr211GlnGly215Pro + Ile220AspArg 64Glu + Leu133IleSer207Glu + Ile220CysAsp 97Glu + Thr206AsnThr106Asp + Tyr137MetSer138Glu + Thr223ProSer138Asp + Ile220AlaSer191Asp + Leu209AsnLeu133Ile + Ser170GluIle107Cys + Ser139GluAsn161Gln + Ser207AspThr206Asn + Ser216GluAsn 67Gln + Ser207AspLeu209Met + Ser219GluGly 68Ser + Asp 97GluGly136Glu + Gly222AsnThr106Pro + Leu209ValGln103Glu + Ile213AlaLeu 96Gln + Ser219GluIle107Asp + Ala164GlnTyr169Asp + Thr211ProLeu133Glu + Leu209ValAsp 97Glu + Asn161SerIle213Gly + Ser216AspThr206Pro + Ser207AspAla164Gln + Leu209CysSer170Glu + Leu209AlaGly160Pro + Leu209CysAsn 67Asp + Asn163GlnThr206Gly + Ser216AspLeu133Thr + Ser216GluAsn163Asp + Leu209ThrVal 95Met + Phe202SerGly136Ser + Gly222SerLeu209Met + Ile220MetGln103Ser + Asn163GluIle107His + Arg218AspPhe192Ala + Gly215GlnLeu209Thr + Ser216GluTyr169Glu + Thr217SerSer101Asp + Leu209AlaLeu209Ala + Ser221AspGly 66Asp + Thr106GlnVal 95Glu + Ile213CysSer105Asp + Thr211GlnAsp165Glu + Asn168SerGly 66Asn + Ile220ThrSer219Glu + Ile220ProGly 68Glu + Ile220MetVal 95His + Leu209MetGly 66Ser + Leu209MetSer140Glu + Gly214GlnGly160Asn + Leu209AspThr206Asp + Ile220MetGly102Asn + Thr206GluGly134Pro + Phe192ProLeu133Asp + Phe202SerAsn162Glu + Ile220HisLeu133Gly + Leu209MetAsn194Ser + Ser219AspGly 70Gln + Thr217ProAsp 97Glu + Thr217GlnGly100Asp + Leu209CysSer216Asp + Ile220ThrArg167Glu + Asn194SerGly 66Gln + Ser170GluSer101Asp + Tyr137AlaAsn 67Gln + Asn161AspAsn168Asp + Leu209IleAsn 67Gln + Gly 68GluAsn161Asp + Thr217ProThr206Asp + Leu209MetAsp 97Glu + Leu209MetAsp 97Glu + Tyr104HisAsp 98Glu + Gly102ProTyr104Ala + Ser191GluGly100Ser + Arg167GluSer207Asp + Leu209SerAsn194Ser + Ser207AspLeu 96Val + Ile220CysAsn163Gln + Ser191AspAsn162Gln + Asp165GluGly 66Gln + Thr 71AsnTyr137Leu + Phe192AsnGly 66Asn + Ser207GluAsp 97Glu + Leu133SerThr206Gln + Ser216AspLeu 96Pro + Ile213GluAsp165Glu + Gly214AsnSer190Glu + Thr217GlyTyr104Met + Ile220GluAsp 65Glu + Thr223SerGly100Asn + Ser170GluGly134Ser + Leu209CysAla164Gly + Ser207AspThr 71Asn + Ile220AspIle107Cys + Ser219AspThr106Gly + Asn162AspAsn161Glu + Leu209ThrGly214Gln + Ser221AspAla164Gln + Ser207GluAsn 67Ser + Ser216AspLeu209Ala + Thr217SerAla166Gln + Ser216AspLeu209Gln + Ile220Glu
表33
多环区三突变变异体Leu 96Gly + Leu209Pro + Ser216GluArg 64Glu + Tyr104Cys + Ile220GlnTyr169Ser + Ser190Asp + Ile220ThrGly100Glu + Asn162Ser + Asn163GlnGln103Glu + Leu133Pro + Leu209AsnGly 70Asp + Leu133Ser + Gly136AsnGly 68Asn + Leu209Gln + Ser216GluAsp 65Glu + Gly135Pro + Ile220AlaGly 66Asn + Asn 67Glu + Leu209SerThr 71Gly + Leu 96Gly + Gly214ProGly 70Gln + Leu209Ile + Ile220GlyTyr169His + Thr217Pro + Ser219AspAsn 67Ser + Val 95Gln + Thr217GluGly136Asn + Gly160Gln + Leu209AspGly136Glu + Asn163Gln + Thr223AsnArg 64Asp + Gly203Asn + Trp212GlnGly 68Ser + Leu209Asn + Gly214ProArg167Asp + Phe192Ile + Thr206AsnSer207Asp + Gly222Ser + Thr223GlyGly160Ser + Asn163Ser + Thr206GlnGly100Asn + Tyr137Ser + Ser219AspAsn 67Asp + Gly100Ser + Tyr137HisSer105Asp + Gly136Asn + Phe192ThrVal 95Thr + Leu133Ser + Asn161GlnSer140Glu + Asn163Ser + Leu209ValPro204Ser + Thr217Glu + Ile220AsnIle107Ser + Ile208Thr + Ser219AspAsp 65Glu + Val 95Ser + Leu209SerAsn 67Gln + Tyr169Gly + Phe202MetGly136Ser + Gly160Pro + Asn162AspVal 95Asn + Ile107Gln + Ser207AspGly135Asp + Thr206Gln + Ile220HisGly 66Pro + Gly100Asn + Ile220ValGly135Pro + Asn162Glu + Ile213ValLeu133Ile + Gly134Pro + Arg167GluGly134Gln + Asn163Ser + Ser216GluAsn194Gln + Ser207Glu + Leu209GlyArg 64Glu + Asn 99Gln + Ile213MetTyr104Ser + Arg167Glu + Ile220AlaVal 95Ala + Gln103Ser + Ser139AspIle107Ala + Gly214Gln + Ile220GluGln103Glu + Gly136Asn + Asn168GlnThr206Pro + Ser216Glu + Ile220CysAsn 67Asp + Ala164Pro + Ile208ThrAsp 65Glu + Asn168Gln + Gly222AsnTyr104Leu + Leu209Ser + Ser219GluIle107Pro + Asn194Glu + Thr206GlyLeu133Asn + Leu209Ile + Ile213AspGly 70Pro + Thr106Gly + Ser219GluAsp 65Glu + Gly102Gln + Tyr169GlyGly100Gln + Leu133Gln + Gly160AspAla164Ser + Ser216Asp + Thr217GlyGly 68Glu + Leu209Ala + Ile220GlnGly 66Glu + Ala164Gln + Gly222GlnThr 71Gly + Phe192Ile + Gly215SerAsn 67Gln + Leu 96Ser + Leu209AspArg 64Glu + Gly 70Ser + Ile220GlnGly 66Ser + Tyr169His + Thr206GlySer170Glu + Thr206Gly + Gly215SerGly 66Ser + Ile107Glu + Leu209AsnSer105Asp + Asn194Gln + Ile220HisLeu 96Met + Asn161Asp + Gly203GlnGly136Gln + Asn161Asp + Phe202IleGly100Gln + Phe192Ala + Ser216AspThr211Pro + Ile220Met + Gly222GlnGly 68Ser + Thr106Gln + Ser207AspAsn161Gln + Thr211Gly + Arg218AspThr106Asn + Asn194Gln + Leu209GlnAsp 65Glu + Tyr169Val + Ile220ValGly 70Asp + Val 95Gly + Leu209MetGly205Ser + Thr206Asp + Ile213ProPhe192Gln + Thr206Glu + Leu209SerGly160Gln + Thr206Gly + Gly214AspGly 70ASn + Arg218Asp + Ile220LeuGly 70Glu + Gly136Asn + Gly203ProIle107Gly + Ile213Gln + Ser219AspGly102Ser + Ala166Pro + Ile220ValTyr104Ile + Gly134Gln + Ile220CysVal 95Pro + Asn162Gln + Ser207AspGly100Gln + Gly136Asn + Asn194GluAsn 99Ser + Thr211Gln + Ile220AspArg 64Glu + Leu133Ile + Ile220SerAsp 97Glu + Thr206Asn + Ile220CysIle107Asn + Thr206Asn + Ser207GluAsn 67Gln + Leu 96Gln + Ile213AspGly 68Ser + Leu209Met + Ser219GluTyr169Asp + Leu209Val + Thr211ProThr206Pro + Ile213Gly + Ser216AspAla164Gln + Ser207Asp + Leu209CysAsn 67Asp + Gly160Pro + Leu209CysAsn163Gln + Thr206Gly + Ser216AspVal 95Met + Phe202Ser + Leu209ThrGly136Ser + Ile220Met + Gly222SerGln103Ser + Asn163Glu + Leu209MetIle107His + Phe192Ala + Arg218AspSer101Asp + Leu209Ala + Thr217SerGly 66Asn + Asp165Glu + Asn168SerGly 66Ser + Leu209Met + Gly214GlnGly134Pro + Ser140Glu + Phe192ProLeu133Gly + Ser207Glu + Leu209MetGly 70Gln + Asn163Asp + Asn194SerGly100Asp + Leu209Cys + Thr217GlnGly 66Gln + Ser101Asp + Tyr137AlaAsn 67Gln + Asn168Asp + Leu209IleAsn161Asp + Leu209Met + Thr217ProGly100Ser + Ser207Asp + Leu209SerLeu 96Val + Asn194Ser + Ser207AspAsn163Gln + Ser191Asp + Ile220CysGly 66Gln + Thr 71Asn + Ser216AspGly 66Asn + Tyr137Leu + Phe192AsnAsp 97Glu + Leu133Ser + Ile220MetLeu 96Pro + Thr206Gln + Ile213GluAsp165Glu + Gly214Asn + Thr217GlyAsp 65Glu + Gly100Asn + Thr223SerGly134Ser + Ser170Glu + Leu209CysThr 71Asn + Ile107Cys + Ser219AspThr106Gly + Asn162Asp + Leu209ThrAsn 67Ser + Ala164Gln + Ser207GluAla166Gln + Leu209Ala + Ser216AspLeu 96Gly + Leu133Ser + Ser190GluSer207Asp + Leu209Thr + Ile220ProTyr137Thr + Ser139Glu + Gly215ProThr206Gly + Leu209Asp + Thr211AsnThr206Asp + Ile208Val + Ile220LeuLeu 96Cys + Ser207Glu + Ile220LeuIle107Leu + Gly136Glu + Ile220GlyThr106Asn + Ser139Glu + Ala164ThrSer191Glu + Leu209Ser + Ile220GlySer140Glu + Gly215Ser + Gly222ProLeu 96Ser + Ala164Gly + Ile220HisAsp 97Glu + Tyr104Asn + Tyr137GlnGly 70Gln + Asp 98Glu + Leu133ThrGly 66Ser + Thr 71Gly + Phe202SerSer101Asp + Thr206Gly + Ile213GlyTyr169Ile + Ser170Glu + Thr211GlyGly160Ser + Ile213Val + Ser219GluLeu209Gly + Ile220Gly + Thr223ProThr106Gln + Ala164Gln + Phe192GluAsn 99Glu + Leu209Gln + Ile220MetGly160Gln + Asn194Glu + Gly215GlnSer105Asp + Phe202Pro + Leu209GlnGly 66Glu + Leu133Thr + Leu209AsnAla164Thr + Leu209Met + Ile220ThrThr 71Gly + Gly214Asp + Gly222AsnVal 95Pro + Thr206Gly + Ile213AspLeu 96Asn + Gly100Asn + Asn194SerArg 64Glu + Gly100Asn + Ile220MetGly 66Ser + Arg218Asp + Gly222SerAsp 65Glu + Asn162Gln + Ile220ThrAsn 99Asp + Thr206Ser + Ile220SerLeu 96Ser + Tyr169His + Ser216AspAsp 98Glu + Tyr169His + Gly215AsnTyr104Leu + Ser207Asp + Leu209SerAsn161Ser + Phe192His + Thr206ProLeu 96Ser + Ser170Asp + Leu209IleThr106Gln + Asn163Gln + Ser207AspGly 70Gln + Asn 99Ser + Asn161SerAsn 99Ser + Ala164Asp + Thr217ProSer139Glu + Thr206Asn + Ile213LeuLeu 96Asn + Thr217Asn + Ile220HisGln103Ser + Leu209Pro + Ser219GluAsp 65Glu + Leu 96Cys + Gln103AsnAsn 67Gln + Ser207Asp + Gly214AsnGly 68Pro + Thr211Ser + Thr223AspTyr137Gln + Ser138Glu + Ile220AsnAsp 65Glu + Phe192Val + Gly215SerThr 71Gln + Gly203Gln + Thr206AspArg 64Asp + Asn 99Ser + Thr106SerGly135Gln + Asn168Ser + Phe192AspLeu133Ala + Leu209Ala + Ser219GluAla164Thr + Asn168Ser + Ile213ProSer207Glu + Gly214Gln + Ile220AsnPhe192Tyr + Leu209Pro + Gly215GlnAsn163Gln + Thr206Gly + Leu209AsnLeu 96Pro + Asp 98Glu + Asn163GlnSer101Asp + Asn168Gln + Tyr169ValGly136Pro + Asn162Glu + Ile220ThrLeu209Cys + Gly214Gln + Ile220CysArg 64Asp + Leu209Ile + Ile220HisGly134Pro + Gly135Ser + Ser219GluAla164Thr + Leu209Thr + Ser219GluTrp212Phe + Ser219Glu + Ile220GluLeu 96Glu + Asp 97Glu + Ile220LeuLeu 96Glu + Asp 97Glu + Gly222ProAsn161Asp + Asn162Glu + Gly214SerGly160Asp + Asn161Glu + Ile220SerTyr137Val + Thr206Glu + Ser207AspGly 68Pro + Arg218Glu + Ser219GluLeu209Thr + Arg218Glu + Ser219AspTyr104Gln + Ser216Asp + Thr217AspLeu209Glu + Thr217Asn + Ser219AspLeu209Asp + Ser219Glu + Ile220SerAsn194Gln + Ser207Asp + Ser221GluAla164Gly + Ser207Asp + Ile220AspLeu 96Val + Ser207Asp + Ile220AspLeu 96Glu + Gly102Glu + Ile213GlnGly 68Asn + Ser207Glu + Ser219GluSer207Asp + Ser219Glu + Ile220CysLeu 96Gln + Ser207Glu + Ser219GluGly102Pro + Ser207Asp + Ser219AspGln103Ser + Ser207Asp + Ser219GluVal 95Gln + Ser207Asp + Ser219AspSer207Asp + Ile213Pro + Ser219GluAsp 65Glu + Asn 67Asp + Ala164HisAsp 65Glu + Asn 67Glu + Gly136SerAsn162Gln + Asp165Glu + Arg167GluLeu209Met + Gly214Asp + Ser216GluSer138Asp + Ser170Asp + Ile220MetSer138Glu + Asn168Gln + Ser170GluArg 64Glu + Gly 66Asp + Thr217ProAsp165Glu + Asn168Asp + Thr211AsnLeu 96Glu + Asp 98Glu + Gly222SerThr206Ser + Ser216Asp + Arg218GluAla164Ser + Ser216Glu + Arg218GluAsn161Asp + Asn162Asp + Thr223AspLeu209Ile + Thr217Asp + Ser219GluSer101Asp + Gln103Asp + Leu133IleAsn 67Glu + Asp 97Glu + Leu209AlaLeu133Ile + Ser190Glu + Gly222GluSer207Asp + Ser219Glu + Gly222AspAla164Asp + Asn168Glu + Leu209ProAsp 65Glu + Gly 68Glu + Asp 97GluAsn163Ser + Thr206Asp + Ser219GluArg 64Glu + Asn 67Asp + Gly215AsnTyr137Asp + Ser139Glu + Arg167AspSer140Asp + Ser170Glu + Gly222SerTyr137Glu + Ser140Asp + Leu209GlnArg 64Glu + Asp 97Glu + Tyr104HisVal 95Glu + Ser105Asp + Thr211GlnGly100Asn + Asn161Glu + Ser221AspAsp 65Glu + Asn 99Glu + Trp212AsnAsp 65Glu + Asn 99Asp + Thr217SerSer190Glu + Ser207Glu + Ile220GluGly 68Glu + Gly136Pro + Ser216GluVal 95Glu + Asp 98Glu + Gly102ProAsn162Asp + Asp165Glu + Ser191GluVal 95Cys + Ser138Asp + Arg167GluLeu209Asp + Ser216Glu + Ile220AlaGly100Glu + Gln103Asp + Ile107GlyGly100Asp + Gly102Pro + Gln103GluSer216Asp + Ser219Glu + Ile220ThrGly102Pro + Ser216Asp + Ser219AspTyr104Gln + Ser216Glu + Ser219GluAsn 99Gln + Ser216Glu + Ser219AspThr206Gln + Ser216Asp + Ser219AspSer207Asp + Ser216Asp + Thr217AspGly134Asp + Asn162Glu + Phe192GluGly102Glu + Ser105Asp + Leu133GluSer216Asp + Ser219Asp + Ser221AspAsn163Asp + Ser170Glu + Asn194AspTyr137Glu + Asn162Glu + Arg167GluSer105Asp + Gly135Asp + Tyr169AspVal 95Asp + Asp 98Glu + Ser105GluGly136Asp + Ser140Asp + Gly203GlnSer207Asp + Ser216Glu + Ile220AspThr106Gly + Tyr137Asp + Asp165GluAsp 65Glu + Gly100Glu + Gln103AspSer101Glu + Arg167Glu + Asn168GluAsn 67Asp + Ser191Asp + Phe192GluAsp 98Glu + Asn 99Glu + Ser216AspAsp 98Glu + Asn 99Asp + Ser216AspArg 64Glu + Asp 65Glu + Asp165GluGly102Glu + Gln103Glu + Ser221GluLeu133Glu + Thr206Asp + Ser207GluSer139Asp + Ser216Asp + Thr217AspAsp165Glu + Gly215Asp + Ser216AspAsp 97Glu + Leu209Glu + Ser219GluSer191Glu + Leu209Glu + Ser219GluThr106Asp + Leu209Asp + Ser219AspAsn 99Glu + Ile213Asp + Ser216GluSer207Glu + Leu209Glu + Ile213AspAsn 67Glu + Asp 98Glu + Leu209AspSer101Asp + Ser207Asp + Ser221AspGly135Asp + Ser207Asp + Ser219GluAsn162Glu + Ser207Asp + Ser219GluSer139Glu + Ser207Glu + Ser219GluGly134Glu + Ser170Glu + Ser216AspAsp165Glu + Arg167Glu + Ser207AspAsp165Glu + Arg167Asp + Ser219AspAsp 65Glu + Ser138Glu + Ser140AspAsp 97Glu + Arg167Glu + Tyr169AspSer207Glu + Ile213Glu + Ser216GluThr 71Pro + Ser191Glu + Ser207GluSer191Asp + Ser207Asp + Gly214GlnGly 66Glu + Gly 70Glu + Ser216GluAsn163Asp + Ser191Asp + Ile220GluAsp 97Glu + Ser207Glu + Leu209GluTyr104Asp + Ser207Glu + Leu209GluSer190Asp + Thr206Glu + Ser216AspAsp 97Glu + Leu133Glu + Asn161SerAsn168Asp + Ser170Asp + Ser190AspAsp 98Glu + Ser221Asp + Thr223GluAsn 67Asp + Asp 97Glu + Gly135GluAsn 67Asp + Asp 97Glu + Ser219AspSer105Glu + Gly160Asp + Gly222AspAsp 98Glu + Gly160Asp + Asn163GluSer139Glu + Ser170Glu + Arg218AspGln103Glu + Ser207Asp + Gly222GluSer207Glu + Ser216Asp + Gly222AspGly135Glu + Gly160Glu + Ser216GluTyr104Glu + Asn163Asp + Ser190GluSer190Glu + Asn194Asp + Ser219AspTyr104Met + Ser190Glu + Ile220GluAsn 67Asp + Ser216Asp + Ser219GluLeu133Asp + Asn162Glu + Phe202SerAsn162Glu + Ser191Asp + Leu209GluAsp 65Glu + Leu 96Asp + Asn162AspArg 64Asp + Gly136Glu + Arg167AspAsn 67Asp + Gly 68Gln + Ser216GluAsn 67Glu + Ser216Glu + Ile220CysSer101Glu + Thr206Asp + Leu209GluAsp 65Glu + Leu209Glu + Ser216GluSer101Glu + Ile107Glu + Ser219GluGly 68Asp + Ser170Glu + Ser216AspGly136Asp + Ser139Glu + Ser216GluGly135Asp + Leu209Ser + Thr223AspLeu133Glu + Asn161Glu + Gly214GluArg 64Glu + Ser191Glu + Ser221AspAsn 67Glu + Gly214Asp + Ser219GluThr106Glu + Ser219Glu + Gly222GluAsp 98Glu + Ile220Asp + Thr223GluArg167Asp + Gly215Glu + Arg218AspTyr104Asp + Ser139Glu + Ser219AspIle107Gly + Asn161Asp + Ile220AspGly160Glu + Ser216Asp + Ser219AspAsp165Glu + Ser216Glu + Ser219AspAsp 98Glu + Ser207Asp + Thr223AspSer139Asp + Asn168Glu + Ser207GluSer105Asp + Ser138Glu + Ile220GluSer105Glu + Ser138Asp + Ser219AspSer105Asp + Ser138Asp + Gly222AspIle213Pro + Ser216Glu + Ile220GluSer101Glu + Gly214Glu + Arg218GluGly136Glu + Asp165Glu + Ser190AspAsp 65Glu + Leu209Cys + Ser216AspSer138Asp + Asn161Glu + Thr206GluGly136Asp + Ser140Asp + Ser219GluGly 68Glu + Gly134Glu + Ser219AspGly 68Asp + Asn194Glu + Ser219AspGly 68Glu + Asn161Glu + Ser219AspGly 68Asp + Ile107Asp + Ser219GluGly 68Glu + Ser139Asp + Ser219AspAsp 98Glu + Gly135Asp + Ser139GluGly135Glu + Ser139Glu + Leu209GluAla164Glu + Ser190Asp + Ser219GluGln103Asp + Tyr137Asp + Gly160SerGly 70Glu + Gly102Asp + Tyr169ProGly 70Glu + Ser219Glu + Gly222AsnGly 70Glu + Leu209Met + Ser221AspIle213Ser + Ser219Asp + Thr223AspGly135Asp + Ser140Glu + Gly215AspGly102Glu + Ser170Glu + Thr206AsnGly102Glu + Gly135Asp + Thr206GluGly 70Glu + Asp165Glu + Ile220AspPhe192Asp + Ser207Asp + Ile213GluAla164Glu + Arg218Asp + Thr223AspGly135Asp + Ser190Glu + Ile220GluAsn 99Glu + Leu133Asp + Asp165GluAsp165Glu + Ser219Glu + Thr223AspAsn 67Glu + Asn162Asp + Ser216Glu
表34
多环区四突变变异体Tyr104Cys + Leu133Ile + Ser216Glu + Ile220GlnGly102Asn + Gly160Ser + Ala164Gly + Arg218AspAsn 67Glu + Thr206Gln + Leu209Met + Ile220MetGln103Glu + Thr106Pro + Leu133Pro + Leu209AsnVal 95His + Gly136Asn + Gly160Gln + Gly222GluArg 64Asp + Gly102Pro + Gly203Asn + Trp212GlnVal 95Thr + Leu133Ser + Asn161Gln + Gly222AsnGly 66Gln + Gly 70Ser + Asn 99Ser + Ser219GluAsp 65Glu + Gly 68Asn + Tyr169Val + Ile220ValPhe192Gln + Gly205Ser + Thr206Glu + Leu209SerLeu133Val + Asn162Asp + Leu209Val + Ile220AlaLeu 96Thr + Leu209Ile + Ile220Val + Thr223SerGly100Glu + Gly102Asn + Gly134Ser + Ile208ProGly100Ser + Asn194Gln + Ser207Glu + Ile220ThrAsn 67Gln + Ala164Gln + Tyr169Asp + Ile220ThrGly160Ser + Ser191Glu + Ile220Ser + Gly222SerThr206Pro + Ser207Asp + Leu209Cys + Ile213GlyVal 95Met + Gly136Ser + Phe202Ser + Gly222SerGln103Ser + Asn163Glu + Leu209Met + Ile220MetIle107His + Phe192Ala + Gly215Gln + Arg218AspGly 66Asn + Asp165Glu + Asn168Ser + Ile220ThrGly100Asn + Ser140Glu + Leu209Met + Gly214GlnGly 70Gln + Asn163Asp + Asn194Ser + Thr217ProAsn161Ser + Phe192His + Thr206Pro + Ser219AspVal 95Cys + Leu 96Ser + Ser138Asp + Leu209IleGly 68Gln + Ser101Asp + Thr106Pro + Ile220MetGly100Pro + Gly102Gln + Leu209Cys + Gly215AspGly100Asn + Gln103Ser + Ser207Asp + Leu209ValThr106Gln + Ile107Asn + Phe192Val + Thr223GlyTyr137Gln + Ser138Asp + Leu209Val + Trp212AsnLeu133Ala + Asn162Asp + Tyr169Val + Leu209IleGly102Ser + Gly134Gln + Gly136Asn + Ser207GluGly100Gln + Gly134Pro + Gly160Asn + Ser219GluTyr137Met + Asn194Gln + Ser207Asp + Gly215ProAla164Asn + Ser190Asp + Phe202Ser + Ile213AsnGly 68Pro + Asp 98Glu + Ala166His + Asn168SerIle107Pro + Gly136Pro + Ser207Glu + Gly222GlnArg 64Asp + Tyr169Gly + Phe192Pro + Gly205SerVal 95Thr + Tyr104Cys + Gly134Asp + Ile208AsnIle107His + Gly135Glu + Phe192Gly + Thr217ProIle107Gly + Ser191Glu + Leu209Met + Thr223GlnGly 68Gln + Val 95Ala + Ser207Glu + Leu209AsnGly 66Asn + Asp 97Glu + Asn161Ser + Gly214AsnTyr137Met + Leu209Ser + Ser216Asp + Ile220ProVal 95Asn + Leu209Val + Ser219Asp + Thr223ProGly135Asn + Ser140Glu + Ala164Thr + Ile220LeuGly 70Ser + Gly135Asp + Thr217Gln + Ile220ThrGly 66Ser + Ser140Asp + Leu209Pro + Ile213LeuVal 95Asn + Leu 96Ile + Thr217Pro + Ile220GlyLeu 96Met + Gly203Asn + Ser219Glu + Ile220GlyTyr104Cys + Tyr169Ser + Leu209Thr + Ser216GluGly 70Gln + Ser138Asp + Thr206Asn + Ile220GlnAsn163Gln + Gly214Pro + Ile220Asn + Thr223GluGly 68Asn + Ser138Asp + Asn168Ser + Ile220MetAsn161Gln + Phe192His + Thr217Asp + Ile220AlaGln103Ser + Phe192Asn + Arg218Glu + Ile220ThrAla164Gly + Asn168Gln + Ser207Asp + Ile208MetThr106Gly + Ser139Asp + Phe192Thr + Gly215AsnLeu209His + Ile213Ser + Gly214Asn + Ser219GluGly 66Asn + Asn 99Ser + Ser101Glu + Asn194GlnTyr137Glu + Thr206Pro + Trp212Thr + Ile220HisGly135Glu + Tyr169Cys + Thr206Gln + Ile220SerThr206Pro + Leu209Ser + Ser216Glu + Ile220ValGly 66Asn + Gly 68Asn + Ser105Asp + Leu209ProSer170Glu + Thr206Gly + Leu209Gln + Ile220CysTyr104Thr + Asn163Ser + Leu209His + Gly222AsnThr106Glu + Tyr137Gln + Asn162Ser + Leu209AlaLeu 96Thr + Ser207Asp + Gly214Ser + Ile220GlyAsn 99Ser + Phe192Pro + Trp212Val + Ser216AspGly136Ser + Tyr137Asp + Leu209Val + Ile220GlyGly 66Ser + Gly203Asn + Ser219Glu + Ile220MetAla164Thr + Thr206Asn + Thr217Ser + Ser219GluGly102Pro + Thr106Asn + Asn161Glu + Tyr169SerThr106Gly + Gly134Gln + Thr206Asn + Ser207GluGly 66Asn + Gln103Asn + Thr106Gln + Ile220GlnArg 64Asp + Asn 67Gln + Ile107Ala + Asn162SerThr 71Gly + Thr106Ser + Asn163Ser + Ile220AsnAsn 67Gln + Ala164Ser + Ser207Asp + Ile220ThrGly 68Ser + Asn163Gln + Arg167Asp + Ile208ThrTyr137Ser + Asn168Glu + Leu209Asn + Ile213AlaGly134Ser + Gly135Pro + Ala164His + Asn168SerAsn163Gln + Ala164Asp + Leu209Gly + Gly215ProGly102Pro + Gly136Pro + Leu209Ser + Ile220ProTyr104Cys + Gly136Asn + Ala164Asn + Leu209HisAsp 65Glu + Leu 96Val + Gly136Asn + Tyr169ProAsn 67Asp + Gly100Pro + Tyr104Leu + Leu209AlaVal 95His + Gly135Gln + Ser191Glu + Ile220AlaAsn163Ser + Asn194Ser + Ser207Glu + Leu209SerGly 66Glu + Leu 96Ile + Tyr104Thr + Ile213AlaGln103Asn + Ser138Glu + Gly160Ser + Phe202CysGly 68Pro + Thr106Ser + Asn194Asp + Thr211SerGly100Gln + Tyr169Thr + Thr211Pro + Thr217SerGly 66Asn + Val 95Cys + Ser139Asp + Asn194GlnGly135Pro + Asn161Glu + Asn162Ser + Leu209AlaGly 66Ser + Thr 71Ser + Val 95Thr + Ile107AsnAsn168Gln + Leu209Pro + Ser219Glu + Ile220GlnGly 66Pro + Tyr137Asn + Tyr169Ile + Thr223AspSer101Glu + Ile107Pro + Leu133Cys + Leu209ProAsn 67Asp + Gly 68Ser + Asn161Ser + Thr223SerAsn 99Gln + Gly102Asn + Ile107Met + Trp212GlyTyr137Ala + Asn194Gln + Ile213Asp + Gly214AsnLeu 96Ala + Leu209Met + Gly222Asn + Thr223GlnLeu 96Val + Asn162Ser + Asn163Gln + Ser170GluAsn 67Ser + Leu133Gln + Leu209Gln + Ser221AspVal 95Gln + Ala164Thr + Ser207Glu + Leu209HisGly100Gln + Asn168Gln + Ser219Asp + Ile220AspAsp 97Glu + Asp 98Glu + Ile213Ala + Thr217GlyGln103Asn + Gly135Ser + Gly215Glu + Ser216GluAsn194Ser + Leu209Gly + Gly215Glu + Ser216GluGly 70Gln + Tyr104Cys + Leu209Glu + Ser219GluIle107Val + Thr206Asp + Ser207Glu + Ser221GluLeu133Gly + Ser207Glu + Leu209Met + Ser221GluVal 95Thr + Tyr137His + Ser207Glu + Ser219GluGly 68Gln + Asn162Ser + Ser207Asp + Ser219GluTyr137Ala + Ser207Glu + Ile213Pro + Ser219AspGly100Asn + Tyr137Ser + Ser207Asp + Ser219AspTyr137Gly + Ser207Glu + Gly215Asn + Ser219GluAsp 65Glu + Asn 67Asp + Phe202Ser + Thr206GlyGly160Ser + Asn161Glu + Asn194Glu + Thr217AsnAsn 67Gln + Ser207Asp + Ser219Asp + Ser221GluGly102Asn + Gly134Gln + Ser138Glu + Ser170AspAsp 65Glu + Gly 68Asp + Gly135Pro + Phe192HisPhe192Asn + Thr206Glu + Ser207Glu + Ser219GluGly160Pro + Thr206Asp + Ser207Glu + Ser219AspLeu 96Asn + Thr206Glu + Ser207Asp + Gly222GluAsp 65Glu + Asp 98Glu + Phe192Leu + Ile213GlnGly 66Gln + Asn162Ser + Ser207Glu + Leu209AspGly 66Ser + Gly136Gln + Ser190Asp + Thr206AspAsn 67Gln + Ile107His + Asp165Glu + Asn168AspPhe202Thr + Ser207Asp + Ile220Glu + Gly222GluAsn 67Ser + Val 95Gln + Leu209Asp + Thr217GluAsn163Asp + Ser191Asp + Asn194Glu + Ile220MetSer207Glu + Leu209Glu + Ile213Met + Ser221AspAsn 67Glu + Leu 96Asp + Asp 97Glu + Leu209AlaThr206Asp + Leu209Asp + Ser219Asp + Ile220ProSer216Glu + Thr217Glu + Ser219Glu + Gly222GlnSer139Glu + Ser170Glu + Phe192His + Leu209GlnGly102Asn + Pro204Ser + Ser207Asp + Gly222GluThr106Gln + Ser207Asp + Leu209Ser + Gly222AspGly100Ser + Ser207Asp + Ile220Asn + Gly222AspThr106Asn + Ser190Glu + Ser207Asp + Ser221GluLeu209Met + Gly214Asp + Thr217Glu + Ile220AsnAla164Gln + Ser207Asp + Arg218Asp + Ile220ValTyr169Gly + Leu209Asp + Ser216Glu + Ser219GluArg167Glu + Asn194Asp + Leu209Ser + Ile220ProGly102Pro + Ile213Asp + Arg218Asp + Ser219GluAsp 98Glu + Gly102Asp + Asn161Gln + Leu209SerIle107Gly + Gly134Asp + Ser138Asp + Tyr169SerGly 68Glu + Gly134Pro + Phe192Pro + Ser216GluAsp 65Glu + Gly214Glu + Ile220Thr + Gly222ProSer190Asp + Ser207Asp + Ile208Met + Ile220ValGly134Asn + Phe202His + Arg218Asp + Ser221AspLeu 96Ala + Ile213Glu + Ser216Glu + Ser219AspGly160Gln + Ser207Asp + Arg218Glu + Gly222AspGly 70Glu + Ile107His + Leu133Glu + Phe192ThrGly136Asp + Ser170Asp + Asn194Asp + Gly222SerTyr104Glu + Leu133Asp + Ile213Ser + Gly215GlnGly100Gln + Ser207Glu + Ser216Asp + Ser219GluGly100Asn + Tyr137Ala + Leu209Asp + Ser216GluAsn162Ser + Ser216Glu + Ser219Asp + Ile220AsnVal 95Ser + Leu 96Pro + Ser216Asp + Ser219AspGly102Pro + Ser216Asp + Ser219Asp + Ile220MetGly135Glu + Gly136Gln + Arg167Asp + Asn194GluAsn 99Gln + Ser207Glu + Ser216Asp + Thr217AspAsp 65Glu + Ser101Glu + Gly102Glu + Leu133ThrAsn163Gln + Ser216Glu + Ser219Asp + Ser221AspGly 66Gln + Asn163Asp + Ser170Glu + Asn194AspGly 66Pro + Gly134Asp + Asn161Glu + Ser191AspLeu133Thr + Asn161Asp + Ser219Asp + Ile220AspSer105Asp + Gly135Asp + Gly136Ser + Tyr169AspAsn 67Gln + Asn163Glu + Asn194Glu + Ser221AspSer207Glu + Ile213Pro + Ser216Glu + Ile220GluGly 68Asp + Leu 96Glu + Thr206Gly + Gly214GluAsn 99Glu + Ser101Glu + Gly136Glu + Asn163GlnSer138Glu + Ser139Asp + Asn163Asp + Ile220MetSer101Glu + Trp212Phe + Ser219Glu + Ile220GluAla164Glu + Asn168Gln + Ser219Glu + Ile220AspSer101Glu + G1y160Gln + Arg167Glu + Asn168GluArg167Glu + Asn168Glu + Asn194Ser + Ser207AspArg167Glu + Asn168Asp + Asn194Ser + Ile220GluLeu 96Thr + Arg167Asp + Asn168Glu + Ser216GluThr106Asp + Leu209Asn + Ser221Asp + Gly222AspAsp 65Glu + Tyr137His + Asn161Glu + Asn162AspAsn 67Asp + Ser191Asp + Phe192Glu + Pro204SerArg 64Asp + Gly 68Ser + Gly222Asp + Thr223GluSer139Glu + Ser140Glu + Arg218Asp + Thr223ProLeu 96Met + Asp 98Glu + Asn 99Asp + Ser219AspArg 64Asp + Asp 65Glu + Gln103Asp + Leu209IleTyr137Glu + Ser138Glu + Ser216Asp + Thr217AsnGly136Asp + Thr206Asp + Ser207Glu + Ile220MetSer101Asp + Ala164Ser + Thr206Asp + Ser207GluGly 66Gln + Tyr104Asp + Ser105Asp + Ser219AspSer138Asp + Thr217Gln + Arg218Glu + Ser219AspArg 64Asp + Leu209Gln + Ser216Glu + Thr217GluSer105Glu + Leu209Asp + Ser219Glu + Thr223GlnSer101Asp + Leu209Asp + Ser219Glu + Ile220SerAsn163Asp + Phe192Leu + Asn194Glu + Ser207AspAsn163Asp + Asn194Glu + Ser207Glu + Trp212HisAla164Asp + Asn194Glu + Ser207Asp + Ile220LeuAla164Glu + Asn194Glu + Ser207Glu + Ile220SerGly100Asp + Ala164Glu + Asn194Asp + Thr206SerLeu 96Pro + Ser207Glu + Ser216Glu + Ser221AspVal 95Asp + Phe192Tyr + Ser207Asp + Ser221AspAla164Asp + Ser207Asp + Gly215Gln + Ser221AspAsp 65Glu + Leu209Gln + Ser216Asp + Arg218AspIle107Cys + Ser139Asp + Ser207Glu + Ser219AspTyr137Glu + Ser207Asp + Ser219Glu + Ile220SerVal 95Gln + Tyr104Asp + Ser207Asp + Ser219AspAsn163Glu + Ser207Glu + Ser219Glu + Gly222SerAsn168Glu + Ser207Glu + Ser219Glu + Gly222AsnGly135Asp + Gly203Ser + Ser207Glu + Ser219GluLeu 96Thr + Ser139Glu + Ser207Asp + Ser219AspGly100Glu + Ser207Asp + Ser219Glu + Ile220CysGln103Glu + Tyr104Pro + Gly136Glu + Ile220AsnGln103Glu + Ser105Glu + Ser219Asp + Ile220CysGln103Glu + Ser105Asp + Tyr137Thr + Thr206GluGly160Asn + Ser207Asp + Gly214Glu + Ser216AspAsp 97Glu + Ser101Asp + Ser207Asp + Ile220ThrThrl06Asp + Tyr137Met + Leu209Glu + Arg218GluGly136Gln + Ser138Asp + Ser170Glu + Ser216GluGly 70Asp + Leu133Ser + Ser138Asp + Ser170AspAsn163Glu + Tyr169Cys + Ser219Asp + Gly222AspArg 64Glu + Gly 66Asp + Asn168Gln + Arg218AspSer105Asp + Gly136Asn + Ser139Asp + Phe192ThrTyr137Leu + Ser191Glu + Ser207Glu + Gly222SerSer191Asp + Ser207Asp + Leu209His + Ile220ThrAsn 99Ser + Ser191Glu + Ser207Asp + Ile220AsnGly 66Asp + Asn 99Asp + Thr106Gln + Ser216AspAsp 97Glu + Phe202Thr + Arg218Glu + Ile220AspGly 70Gln + Asn 99Asp + Ser207Glu + Leu209GluThr106Glu + Ser207Glu + Leu209Asp + Gly214GlnAsp 65Glu + Tyr137Pro + Asp165Glu + Asn168AspAsn 99Gln + Asp165Glu + Asn168Asp + Gly215GluAsn 67Glu + Asn161Glu + Ile220Ala + Gly222GluGly 66Asp + Leu 96Gly + Leu209Glu + Ile220AspAsp 98Glu + Leu209Ala + Ser219Glu + Ser221AspAsn 67Glu + Ala164Gln + Ser219Glu + Ser221AspGlyl00Glu + Gly136Asn + Ser216Asp + Arg218GluIle107Asp + Thr206Ser + Ser216Asp + Arg218GluAla164Gly + Asn194Asp + Thr217Asp + Ser219GluAsp 98Glu + Asn163Asp + Asp165Glu + Trp212TyrSer139Glu + Gly215Glu + Thr217Asp + Gly222ProSer101Glu + Ser105Glu + Phe192Val + Leu209HisTyr104Asn + Leu209Glu + Ser216Asp + Gly222AspGly 70Glu + Tyr104Ala + Thr206Asp + Ser219GluLeu 96Glu + Asn 99Asp + Ala164Gln + Gly214GluSer139Asp + Asn161Glu + Ala164Glu + Ile220GlyAsn 67Ser + Gly 70Asp + Leu 96Glu + Ser221GluAsn 67Asp + Asp 97Glu + Asn163Ser + Ser221GluSer170Glu + Ser207Asp + Ile220Ser + Gly222AspLeu 96Gly + Asn163Asp + Thr206Asp + Ser219GluAsn 67Asp + Ser190Asp + Gly215Ser + Gly222AspTyr137Cys + Arg167Asp + Ser170Asp + Ser219AspGly135Glu + Ser138Asp + Gly214Glu + Gly222SerAsp 65Glu + Asp 97Glu + Asn194Glu + Gly205AsnGly102Ser + Serl05Asp + Leu133Glu + Thr223AspGly 70Asp + Asp 98Glu + Leu209Met + Ser216AspSer138Glu + Ala164Ser + Thr206Glu + Ser219GluGly 68Pro + Asp 97Glu + Thr206Asp + Ser219AspSer105Glu + Ser140Glu + Arg218Glu + Thr223GlnAsn 99Asp + Gly102Glu + Ile213Val + Ser219GluAsn 99Asp + Gly102Asp + Gly136Ser + Ser138AspGly 70Glu + Val 95Glu + Gly100Asn + Gly222GluAsn 67Glu + Ser216Glu + Ser219Asp + Ile220CysAsn 67Asp + Ala164Pro + Ser216Glu + Ser219AspAsn 99Asp + Asn161Glu + Ser190Asp + Gly215AsnVal 95Asp + Asnl61Gln + Ser190Glu + Ser221AspGly 68Asp + Gly100Glu + Gly222Ser + Thr223GluGly134Glu + Tyr169Leu + Ser207Glu + Arg218GluThr106Asp + Ser207Glu + Arg218Asp + Ile220CysArg 64Glu + Asn163Ser + Ser207Asp + Arg218AspSer139Asp + Ser207Asp + Arg218Asp + Ile220ThrLeu133Asp + Gly134Pro + Asn162Asp + Tyr169AsnVal 95Asp + Gly134Glu + Asn194Glu + Thr211GlyIle107Asn + Ser140Glu + Ser170Glu + Ser219AspTyr137Cys + Ser140Glu + Ser170Asp + Ser219GluTyr137Thr + Ser140Glu + Ser170Glu + Thr206GluTyr137Asn + Gly160Glu + Asp165Glu + Ser216GluAsn 99Glu + Gly160Asp + Asp165Glu + Leu209GlnAsp 97Glu + Tyr104Glu + Leu133Ser + Ile220LeuAsn 67Glu + Val 95Gly + Pro204Ser + Ser216AspAsn 99Asp + Asn162Gln + Ser191Asp + Gly222GluGly100Glu + Thr106Gly + Asn162Asp + Tyr169GluAsp 65Glu + Asn 99Asp + Gly135Asn + Gly160AspAsp 65Glu + Asn 99Glu + Trp212Asn + Ser219AspGly134Glu + Asn161Asp + Thr217Pro + Ser219AspAsp 97Glu + Gln103Glu + Asn163Gln + Ile220GluAsn161Glu + Asp165Glu + Ser207Asp + Thr223GlnSer140Glu + Ala164His + Thr217Asp + Ile220AspGly 70Ser + Gly102Asp + Leu133Asp + Ser191AspSer101Glu + Ile107Glu + Thr217Pro + Ser219GluTyr137Glu + Asn162Glu + Ser190Asp + Ile213GlnGly 68Asp + Ser170Glu + Ser216Asp + Ile220ValGln103Asp + Gly135Asp + Leu209Met + Ser216AspTyr104Val + Gly136Glu + Ser139Glu + Ser190AspGly102Pro + Asn161Glu + Asp165Glu + Gly214GluGly 70Glu + Tyr169Cys + Ser190Glu + Ser207GluAsn168Glu + Ser190Glu + Pro204Ser + Ser207GluSer190Asp + Ser207Asp + Leu209Ser + Ser216GluAsn 67Glu + Ser101Glu + Tyr137Asp + Gly215GlnArg 64Glu + Ser191Glu + Ile220His + Ser221AspArg167Asp + Ser191Asp + Ile213Ala + Ser221GluArg167Glu + Thr206Ser + Ser219Asp + Gly222GluSer101Glu + Asn161Gln + Asn162Asp + Ser190AspSer101Asp + Gly136Pro + Ser216Asp + Ser219GluTyr104Gln + Thr106Glu + Ser216Glu + Ser219GluGly 68Pro + Leu133Glu + Ser216Glu + Ser219GluGly160Glu + Leu209Val + Ser216Glu + Ser219GluSer191Glu + Leu209Gln + Ser216Asp + Ser219AspAsn 67Gln + Arg167Glu + Ser216Glu + Ser219AspAsn 99Gln + Ser170Asp + Ser216Glu + Ser219AspSer138Glu + Asn162Gln + Ser216Glu + Ser219GluGly135Gln + Asn168Glu + Ser216Asp + Ser219AspGly100Glu + Thr206Ser + Ser207Glu + Thr223GluAsn163Gln + Gly214Asn + Ser216Glu + Ile220AspGly100Pro + Thr211Asn + Ser216Asp + Ile220GluThr206Pro + Leu209Thr + Ser216Glu + Ile220AspGlyl02Asp + Gly160Asp + Ser190Glu + Trp212SerLeu133Cys + Gly134Asp + Asn162Asp + Ser216AspAsp 65Glu + Thr106Gln + Ser216Glu + Gly222GlnAsp 98Glu + Gly100Ser + Asn168Asp + Ile213GluSer101Asp + Tyr137Ala + Asn161Glu + Thr206GluGly 70Asp + Gly136Glu + Ser140Glu + Ile220ProGly 68Glu + Tyr104Glu + Leu209Val + Ser219AspGly 68Asp + Gly160Gln + Asn194Glu + Ser219AspAsn163Gln + Ser190Glu + Ser216Glu + Ile220GluArg 64Asp + Gln103Asn + Leu133Asp + Gly214GluArg 64Glu + Tyr104Ser + Leu209Glu + Gly214AspGly 70Asp + Ser101Asp + Ser140Glu + Ile220ValGly 70Asp + Leu 96Thr + Leu209Asn + Ser216GluIle107Asp + Ser138Glu + Ala166Gln + Ser219AspAsn161Glu + Ser207Asp + Leu209Ile + Thr217AspGly 68Asn + Asn163Glu + Arg167Glu + Ser219AspAsn 99Ser + Gln103Asp + Gly136Glu + Thr206GluGln103Glu + Leu133Gln + Gly136Glu + Ser216GluGlyl02Asp + Leu133Thr + Ser207Asp + Thr217GluAsp 97Glu + Ser207Asp + Thr217Glu + Thr223AsnSer101Glu + Ser191Asp + Ser207Glu + Leu209CysIle107Ser + Ser191Glu + Ser207Asp + Ser216AspThr 71Pro + Ser191Glu + Ser207Glu + Ser216GluAlal64Gln + Ser191Glu + Ser207Glu + Ser216AspSer170Glu + Asn194Asp + Ser207Glu + Ile220ValSer170Asp + Asn194Glu + Trp212Ser + Ser216GluArg 64Glu + Asn 99Glu + Ser170Asp + Leu209ThrAsp 97Glu + Leu133Glu + Asn161Ser + Ser216AspTyr137Pro + Ser140Asp + Phe192Asp + Ser207GluAsn 99Ser + Gln103Glu + Ser170Asp + Ser219AspGln103Glu + Asn163Glu + Ser170Glu + Ile220HisAsp 65Glu + Ash 99Ser + Ser101Glu + Ser191AspSer105Glu + Gly135Asp + Thr206Asp + Thr217GlnLeu 96Cys + Asn161Glu + Arg167Glu + Arg218AspArg 64Glu + Gly100Glu + Asn161Ser + Leu209GluVal 95Met + Asp 97Glu + Ser105Glu + Ile220AsnGly 68Glu + Val 95Glu + Asn168Ser + Leu209AlaAsn 67Glu + Asn161Asp + Ser216Asp + Gly222AsnAsn 67Asp + Leu133His + Arg167Glu + Ser216GluSer105Asp + Gly160Ser + Ser170Asp + Thr206GluGly 66Pro + Asn162Asp + Ser170Glu + Ser219GluGly100Glu + Gly102Asn + Tyr104Asp + Ser207AspGly100Glu + Tyr104Asp + Gly160Glu + Gly215Gln
表35
多环区五突变变异体Leu133Gln + Gly136Glu + Thr206Gln + Leu209Met +
Ile220MetGly203Asn + Gly205Gln + Ser216Glu + Thr217Gly +
Ile220GlyVal 95His + Ala164Ser + Ser170Glu + Phe192Met +
Thr223GlyLeu133Asn + Asn161Gln + Asn163Gln + Leu209Met +
Ile220LeuAsn 67Gln + Gly136Ser + Asn162Asp + Tyr169Gly +
Phe202MetIle107Asn + Leu209Gln + Ile213Gly + Ser219Glu +
Ile220HisGly 66Ser + Thr 71Gly + Tyr169His + Phe202Ser +
Ile220AspGly135Ser + Ala166Ser + Ser207Glu + Ile213Gly +
Gly222SerAsp 98Glu + Thr106Pro + Tyr137Cys + Trp212Ile +
Ile220ValGly134Asn + Ala166Pro + Phe192Met + Thr206Gly +
Ser219GluThr106Glu + Gly205Asn + Leu209Cys + Gly215Pro +
Ile220ThrAsn163Gln + Asn168Gln + Phe192Cys + Thr206Gln +
Leu209SerLeu 96Gly + Ile107Gly + Ser140Asp + Leu209Ala +
Thr223GlyAsn 67Ser + Val 95His + Gly135Gln + Ser191Glu +
Ile220AlaIle107Gly + Tyr169Ser + Asn194Ser + Ser207Glu +
Leu209SerTyr137His + Leu209Met + Ile220Val + Gly222Asn +
Thr223GlnVal 95Ala + Tyr137Pro + Ala164Glu + Asn168Gln +
Thr206AsnLeu 96Gln + Tyr137Leu + Tyr169His + Leu209Asp +
Ile220ProGly 66Pro + Gly 70Asn + Leu133Pro + Phe202Pro +
Ser207GluVal 95Ala + Leu 96Met + Tyr104Asn + Ile107Ala +
Ser216AspGly 70Ser + Ile107Gly + Leu133Met + Phe192His +
Asn194AspGly 68Pro + Val 95Ser + Gly100Asn + Tyr137Cys +
Ile220ProGly 66Ser + Gly102Gln + Ala164Gln + Ser219Glu +
Gly222AsnGly135Gln + Ser190Glu + Leu209Cys + Gly215Gln +
Ile220ValGly 68Pro + Asn163Asp + Phe192Pro + Leu209Gly +
Ile220ThrAsn 67Gln + Gly 70Asn + Ile107Leu + Gly136Asn +
Asn162GluGly 70Pro + Ile107His + Ser190Glu + Pro204Asn +
Leu209GlnLeu 96Ile + Asp165Glu + Phe202Gly + Leu209Asn +
Thr211GlyGly 66Pro + Gly 70Asp + Tyr169Asn + Leu209His +
Gly215SerTyr104Met + Asn194Glu + Leu209Cys + Thr217Pro +
Ile220GlnGly 70Asn + Leu 96Ile + Tyr104His + Asn161Asp +
Ile220GlnVal 95Thr + Gly102Pro + Thr206Ser + Gly214Glu +
Gly222SerThr106Gln + Asn163Ser + Ile213Pro + Ser216Glu +
Ile220SerLeu 96Cys + Gly136Asn + Ser170Glu + Thr206Gln +
Ile220GlnGly 66Gln + Ser105Glu + Gly135Pro + Gly205Asn +
Thr206SerGly102Pro + Tyr104Ile + Thr211Gly + Ser216Glu +
Thr223GlnTyr104Ala + Gly203Asn + Leu209Gly + Arg218Asp +
Ile220CysGly 66Pro + Val 95Thr + Asn161Ser + Asn162Gln +
Ile220CysGly102Asn + Gly136Gln + Phe202Met + Ile208Val +
Ile213GluGly 66Ser + Gly 68Ser + Gly134Gln + Ala164Gly +
Asp165GluGly 66Pro + Ala164Asp + Tyr169Ser + Thr206Gln +
Thr217AsnLeu 96Ile + Gly136Gln + Asn161Ser + Thr206Gln +
Ser207AspAsn 67Asp + Tyr104Asn + Gly135Gln + Tyr169Ile +
Leu209HisGly 70Pro + Gly136Pro + Ser190Asp + Leu209Ser +
Gly214ProAsp 65Glu + Tyr137Val + Ala164Asn + Phe192Ala +
Thr206SerLeu 96Thr + Gln103Ser + Tyr104Asn + Asp165Glu +
Ile220HisGly 68Asp + Gly 70Pro + Asn 99Ser + Gly214Ser +
Thr223SerLeu 96Ser + Leu133Ala + Tyr137Met + Leu209Ile +
Ser221AspGly134Gln + Asn194Gln + Thr206Ser + Leu209Met +
Ile213LeuGly 70Glu + Leu133Cys + Gly135Gln + Thr206Gly +
Gly222AsnGly134Ser + Ala166Gln + Tyr169Cys + Thr206Gly +
Ser221AspGly160Ser + Thr206Asp + Ile213Gln + Ile220Pro +
Thr223GlyTyr104Asn + Gly135Pro + Leu209Ile + Ser219Glu +
Ile220ValAsn 67Gln + Thrl06Asn + Gly160Glu + Tyr169Asn +
Leu209AlaVal 95Glu + Gly100Pro + Thr206Asn + Leu209Thr +
Ile220AlaAsp 98Glu + Gly102Ser + Ala164Gln + Thr206Ser +
Ile220AsnGln103Asn + Gly136Pro + Ser138Asp + Leu209Gly +
Ile220GlnAsn 67Gln + Thr106Asn + Gly136Gln + Thr206Ser +
Ile220AsnAsn 67Gln + Gly134Gln + Tyr169Cys + Ser219Asp +
Ile220AspAla166His + Thr206Glu + Ser207Asp + Ile213Cys +
Ile220HisGly 70Pro + Ile107Gly + Leu209Gln + Ile220Glu +
Ser221AspAsn 99Ser + Gly160Ser + Gly215Glu + Ser216Glu +
Thr217GlnGly102Gln + Ile107Cys + Ala164Ser + Leu209Asp +
Ser219AspGly134Asn + Tyr137Gln + Leu209Glu + Ser219Asp +
Ile220MetGly102Gln + Gly160Gln + Leu209Asp + Arg218Asp +
Ser219GluGly 70Asn + Ser207Glu + Leu209Val + Ser219Glu +
Ile220GluAla164Asn + Ser207Asp + Thr217Pro + Ser219Glu +
Ile220AspVal 95Thr + Ala164Pro + Ser207Glu + Ile213Val +
Ser221Asp
Val 95Asn + Gly102Pro + Gly160Pro + Ser207Asp +
Ser219AspAsn 67Gln + Ser207Glu + Leu209Val + Ser219Asp +
Ile220AlaAsn 67Gln + Gly136Gln + Ser138Glu + Ser140Asp +
Leu209MetTyr104Asn + Ser138Glu + Ser140Asp + Leu209Thr +
Ile220ValAsp 97Glu + Asn 99Asp + Gly102Gln + Tyr137Ser +
Ile220CysAsn161Asp + Asn163Gln + Tyr169Ala + Asn194Asp +
Leu209AlaThr206Glu + Ser207Asp + Leu209His + Ser219Asp +
Thr223GlyGly102Ser + Gly136Asn + Thr206Glu + Leu209Thr +
Gly222AspThr 71Gly + Gly102Asn + Ser219Asp + Ile220Cys +
Ser221GluThr106Ser + Gly160Asn + Gly205Asn + Ser219Asp +
Ser221AspVal 95Gln + Leu133Gln + Gly215Asn + Thr217Asp +
Ser219AspLeu209Ser + Ser219Glu + Ile220Gly + Ser221Glu +
Gly222AspGln103Ser + Gly203Pro + Ser216Glu + Arg218Glu +
Ser219AspVal 95Ser + Gly135Gln + Ser138Asp + Tyr169Glu +
Ile213ThrAsn 67Gln + Gly160Pro + Ser207Glu + Leu209Cys +
Gly222GluGly135Asp + Ser138Glu + Gly203Gln + Ile213Met +
Gly222AsnAsn162Gln + Ser207Asp + Leu209Pro + Ser219Glu +
Gly222AspTyr137Asn + Ala164Asp + Arg167Asp + Leu209Ile +
Ile220MetLeu 96Gln + Gly102Asn + Gly135Asp + Asn168Asp +
Ile220AlaVal 95Pro + Asn 99Asp + Gly102Asp + Gly160Gln +
Leu209AsnAsn 99Ser + Ile107Gly + Asn162Gln + Leu209Asp +
Ser221GluGly 66Pro + Asn162Asp + Ser191Asp + Thr206Asn +
Thr223AsnGln103Asp + Tyr104Asn + Ser105Glu + Ser140Asp +
Leu209CysLeu 96Ser + Gly136Ser + Asn168Glu + Asn194Asp +
Thr206AsnGly 68Glu + Val 95Ala + Gly136Asn + Gly203Asn +
Ser216AspAsp 65Glu + Thr106Asn + Gly134Gln + Gly214Asp +
Ser216GluGly 68Gln + Val 95Pro + Asn162Gln + Ser190Glu +
Ser207AspAla166Thr + Ser190Glu + Ser207Glu + Leu209Gln +
Thr217GlyGly100Pro + Gly160Gln + Ser190Glu + Ser207Asp +
Leu209ProAsn 67Glu + Ser101Glu + Tyr104His + Phe192Ser +
Leu209ThrSer191Glu + Phe202His + Leu209Met + Ser221Asp +
Thr223GlnGly134Asp + Ser139Asp + Ser140Glu + Ile213Met +
Thr223AsnTyr137Asn + Tyr169Ile + Thr206Asp + Ser219Asp +
Thr223AspTyr104Cys + Leu133Ile + Ser207Glu + Ser216Glu +
Arg218AspGly 68Asn + Tyr137Ser + Ser207Glu + Ser216Asp +
Ser219GluTyr137Ser + Asn168Ser + Gly214Asp + Ser216Asp +
Ser219GluIle107His + Asn162Ser + Ser216Glu + Ser219Asp +
Ile220AsnAsn 67Gln + Asn163Ser + Ser216Asp + Ser219Glu +
Thr223SerLeu 96Asn + Leu133Pro + Thr206Gln + Ser216Asp +
Ser219AspSer191Glu + Thr206Asp + Gly214Asn + Ser219Asp +
Thr223AsnGly 66Ser + Ser105Glu + Thr106Pro + Ser138Glu +
Leu209IleGly136Asp + Gly160Glu + Asn163Ser + Ile220Ser +
Gly222AspIle107Met + Asn162Asp + Ser190Asp + Ser221Asp +
Gly222SerGly 70Gln + Gly214Ser + Ser216Glu + Ser219Glu +
Ser221GluGly136Glu + Asn162Glu + Gly214Ser + Gly215Asn +
Ile220LeuGly 68Asp + Leu209Thr + Arg218Glu + Ser221Asp +
Thr223ProGly100Ser + Phe192Glu + Gly215Asn + Ser219Asp +
Ile220GluGly134Gln + Phe192Glu + Ile213Gly + Ser219Asp +
Ile220GluThr106Gly + Asn162Ser + Asn194Asp + Thr206Asp +
Ser221AspGln103Asn + Asnl61Asp + Ser207Asp + Thr217Ser +
Ile220GluGly 68Asn + Phe192Asp + Ser207Asp + Leu209Asp +
Ile220AsnGly 68Asp + Phe192Tyr + Leu209Gly + Ser219Asp +
Ile220GlnArg 64Asp + Asp 98Glu + Tyr137Val + Phe192Val +
Gly214AspTyr104Gly + Ile107Glu + Gly160Glu + Ser170Glu +
Leu209CysVal 95Thr + Ile107Gly + Ser191Glu + Ser219Glu +
Ile220AspAsp 65Glu + Gly 66Asp + Gly100Asn + Gly102Ser +
Ser190GluAsp 65Glu + Gly 66Asp + Gly134Gln + Ile220Asp +
Thr223GlnAsp 65Glu + Gly 66Asp + Leu209Glu + Ile213Ala +
Ile220AlaAsn 99Gln + Serl90Glu + Serl91Asp + Ser219Glu +
Ile220HisGly 70Asp + Ser101Asp + Tyr137Cys + Gly160Ser +
Ile220HisTyr137Ser + Ser138Asp + Ser139Glu + Ile213Met +
Ser216AspGly135Ser + Ser138Glu + Asn194Ser + Ser219Glu +
Ile220GluThr 71Pro + Asp 97Glu + Thr106Ser + Ser219Glu +
Ile220GluArg 64Asp + Gly 66Gln + Thr206Gly + Ser219Glu +
Ile220AspVal 95Ala + Gln103Ser + Ser139Asp + Arg167Glu +
Ile220AlaSer101Asp + Gly102Glu + Ile213Val + Thr217Ser +
Thr223GluAsn 99Asp + Gly100Glu + Tyr137Thr + Asn163Glu +
Gly215GlnGly 66Ser + Ile107Glu + Leu209Asn + Ser221Glu +
Gly222GluAsn 67Glu + Thr 71Gln + Asnl61Asp + Asn162Glu +
Gly214SerGly100Asp + Ser101Glu + Thr106Gln + Ile213Cys +
Ser221AspArg 64Asp + Asp 65Glu + Gly 68Ser + Leu 96Gln +
Asn168AspGly160Pro + Thr206Glu + Ser207Glu + Gly215Glu +
Gly222ProGly102Ser + Tyr137Asp + Tyr169His + Thr206Glu +
Ser207AspGly 70Asp + Asn 99Ser + Tyr104Glu + Ser105Asp +
Thr217GlnLeu 96Glu + Gly135Ser + Thr217Ser + Ile220Glu +
Ser221GluAsp 97Glu + Asp 98Glu + Gly205Pro + Gly215Ser +
Ser219GluGly 68Gln + Ser138Asp + Leu209Ile + Arg218Asp +
Ser219AspAsn 67Ser + Gly136Asp + Gly215Pro + Ser216Glu +
Thr217AspAsn 99Gln + Ser101Glu + Asn161Gln + Asn162Asp +
Asn163AspTyr137Gln + Tyr169Thr + Ser207Asp + Gly215Asp +
Ser216AspLeu 96Cys + Ser190Asp + Gly215Glu + Ser216Glu +
Ile220CysGly160Pro + Phe192Asn + Ser207Glu + Gly215Asp +
Ser216GluThr 71Asn + Phe192Val + Ser207Asp + Gly215Asp +
Ser216AspGly134Glu + Asn168Asp + Trp212Ile + Ile220Asn +
Thr223GluGln103Asp + Gly134Ser + Ser139Glu + Asn162Ser +
Ser170AspGln103Asp + Thr106Ser + Ser138Glu + Leu209Val +
Gly215GlnASn 67Gln + Gln103Asp + Gly135Gln + Ser138Glu +
Leu209AsnAsp 97Glu + Ile107Gln + Gly136Ser + Ser207Asp +
Ser221AspTyr137Ile + Ser138Glu + Asn194Ser + Ser207Asp +
Ser221AspGln103Asp + Gly135Asn + Gly136Pro + Ser207Glu +
Ile220GluGly102Gln + Gly135Glu + Gly203Ser + ser207Glu +
Ile220GluTyr104Ile + Asn163Asp + Arg167Glu + Tyr169Cys +
Ile208CysGly 66Glu + Asp 97Glu + Tyr104Glu + Gly136Gln +
Leu209MetGly102Ser + Ile107Cys + Arg167Asp + Ser207Asp +
Ser219AspGly100Asn + Gly134Asp + Ser207Glu + Gly214Ser +
Ser219AspArg 64Asp + Val 95Ser + Thr206Ser + Ser207Asp +
Ser219GluAsn194Asp + Ser207Glu + Ser219Asp + Ile220Met +
Thr223AsnSerl40Glu + Ser207Glu + Leu209Ala + Gly215Gln +
Ser219GluTyr104Ser + Gly160Glu + Ser207Glu + Gly215Ser +
Ser219AspAsp 65Glu + Thr206Ser + Ser207Glu + Gly215Ser +
Ser219AspGln103Ser + Asn168Glu + Pro204Gln + Ser207Glu +
Ser219AspLeu133Thr + Ser140Glu + Asn161Ser + Ser207Asp +
Ser219GluLeu 96Pro + Ser140Glu + Ser207Glu + Leu209Thr +
Ser219GluAsn161Asp + Asn168Gln + Ser170Asp + Ser191Asp +
Phe192GlnGln103Glu + Thr106Glu + Leu209Gly + Ile213His +
Ser219GluAsp 65Glu + Asn 67Asp + Thr106Asn + Asn194Glu +
Ile208AsnAsp 65Glu + Asn 67Glu + Leu 96Ala + Gly102Ser +
Tyr169AspAsn 99Ser + Asp165Glu + Arg167Asp + Ile213Gly +
Ser219AspTyr104Ile + Tyr137Ile + Asp165Glu + Arg167Glu +
Ser219AspSer190Glu + Phe192Glu + Leu209Cys + Ser219Glu +
Ile220AlaSer138Glu + Ser140Glu + Thr206Gly + Ile220His +
Ser221GluVal 95Pro + Ile107Ser + Ser138Asp + Ser140Glu +
Ser219AspAsp 65Glu + Gly102Ser + Ser138Glu + Ser140Asp +
Ala166GlyVal 95Cys + Ser101Glu + Ser138Glu + Ser140Asp +
Phe192SerAsp 97Glu + Asn 99Asp + Ser139Asp + Ile213Thr +
Thr223GlyAsp 97Glu + Asn 99Asp + Ser140Glu + Ala166Gln +
Phe192ThrLeu 96Met + Gly136Glu + Asn161Asp + Asn168Gln +
Ile220ProGln103Asp + Ser105Glu + Asn162Ser + Ser207Glu +
Thr223GlnArg 64Glu + Gln103Glu + Ser105Asp + Asn163Gln +
Leu209CysIyr137Ala + Ser170Glu + Phe192Met + Gly214Glu +
Ser216AspGly 70Ser + Ser105Glu + Gly214Glu + Ser216Glu +
Thr223AsnGln103Ser + Asn168Asp + Gly205Ser + Gly214Asp +
Ser216AspGly135Glu + Tyr169Glu + Ser207Asp + Leu209Met +
Ile220CysAsn 99Gln + Gly136Asp + Gly160Ser + Ser170Asp +
Ser216AspGly136Asp + Tyr169Ser + Ser170Asp + Ile213Glu +
Thr217AsnAsp 97Glu + Ser101Asp + Ser139Glu + Asn162Ser +
Ile220ValAsp 97Glu + Ser101Glu + Ile213Met + Thr217Gly +
Arg218AspThr106Asn + Ile107Pro + Asn194Glu + Ser219Asp +
Ser221GluLeu 96Met + Asn 99Asp + Ser101Glu + Ser139Asp +
Leu209ThrAsn 99Asp + Ser101Glu + Gln103Ser + Ser139Asp +
Ile220ProGly 68Glu + Asp 97Glu + Gly160Asn + Asn163Ser +
Ile220AspArg 64Asp + Gly 66Asp + Ile107Gly + Ser207Glu +
Ile220ValGly 70Gln + Gln103Asp + Leu133Ile + Ser140Asp +
Leu209ThrAsn 67Glu + Gly 70Glu + Asn 99Gln + Tyr104Met +
Leu209AspTyr104Gln + Ile107His + Ser191Asp + Thr206Glu +
Arg218AspAsn161Glu + Ala164Gln + Ile213Asp + Ser216Asp +
Thr217AsnThr106Gln + Gly136Pro + Thr206Ser + Ser207Glu +
Thr217GluAsp 65Glu + Leu 96Gly + Asp 98Glu + Thr106Asp +
Ile220GlnAsp 65Glu + Asp 98Glu + Ile107Gly + Gly135Asn +
Gly136AspGly136Asp + Ser138Asp + Ser207Glu + Leu209Asn +
Gly222AsnThr106Gln + Gly136Asp + Ser138Asp + Leu209Cys +
Ser216GluVal 95Ala + Asp 97Glu + Asn168Ser + Ser207Asp +
Leu209GluIle107Glu + Thr206Ser + Ser207Glu + Leu209Glu +
Thr211GlyGly 70Pro + Leu 96Val + Gly102Glu + Ser207Glu +
Leu209AspVal 95His + Asn194Asp + Ser207Asp + Leu209Asp +
Thr217AsnSer140Asp + Ala164Pro + Ser207Asp + Leu209Asp +
Ile220GlyGln103Asp + Tyr104Gln + Gly160Ser + Asp165Glu +
Asn168AspAsp 65Glu + Gly 66Ser + Gly 70Glu + Gly136Asn +
Ser207AspGly 68Asp + Phe192Asn + Gly205Asn + Thr217Asp +
Ser221AspGly134Asp + Gly136Asp + Ser207Glu + Leu209Ile +
Ile220CysGly100Asn + Gly136Glu + Gly160Gln + Ser219Asp +
Ser221GluGly102Ser + Ser170Glu + Gly215Asn + Ser219Glu +
Ser221GluGly 66Ser + Asn163Gln + Arg167Glu + Ser219Glu +
Ser221AspGly100Glu + Gly136Asn + Leu209Ser + Ser219Glu +
Ser221AspLeu133Ala + Gly134Asp + Ile213Cys + Ser219Glu +
Ser221GluGly102Asn + Gln103Glu + Thr206Ser + Ser216Glu +
Arg218AspAsp 98Glu + Thr206Gly + Ile213Ala + Ser216Asp +
Arg218GluAsn163Asp + Ile208Asn + Ile213Asn + Ser216Asp +
Arg218AspGly 66Ser + Gly 70Glu + Tyr169His + Thr206Gly +
Ile220AspArg 64Glu + Asn 67Glu + Ile107G1y + Leu209Val +
Ile220GluGln103Glu + Gly134Ser + Ser138Glu + Arg167Asp +
Thr223AsnGly100Pro + Tyr104Pro + Leu133Gln + Phe192Asp +
Ser207GluGly 68Asn + Ser138Glu + Asn163Glu + Arg167Asp +
Thr206GlyGly 70Ser + Gln103Asp + Asn162Ser + Ser170Asp +
Leu209MetAsp 98Glu + Gly100Ser + Ser101Asp + Asn161Ser +
Ile220AspTyr137Pro + Ser139Asp + Asn161Glu + Ala164Glu +
Ile220GlyGly 66Asp + Asn 99Glu + Tyr104Met + Ser207Glu +
Leu209ProGly 66Asp + Asp 97Glu + Tyr104Gln + Ile208Val +
Leu209AspIle107Asp + Phe192Met + Ser207Asp + Gly222Glu +
Thr223GlnArg 64Glu + Leu 96Ala + Ala164Asn + Ser207Glu +
Gly222AspLeu 96His + Ser207Glu + Ser216Glu + Thr217Asn +
Gly222GluTyr169His + Ser170Glu + Ser207Asp + Ile220Ser +
Gly222AspGly 68Pro + Leu133Glu + Leu209Asp + Gly214Gln +
Ser221GluSer101Asp + Arg167Asp + Ser170Glu + Gly215Asn +
Thr223GlyAsp 65Glu + Asp 97Glu + Ser207Asp + Gly222Ser +
Thr223SerAsp 65Glu + Asp 97Glu + Asn 99Gln + Gly135Glu +
Thr206GlyTyr104Met + Gly160Asp + Ala164Glu + Leu209Asp +
Ile220ThrLeu 96Ser + Asn162Glu + Asp165Glu + Tyr169Cys +
Arg218GluAsp 65Glu + Ile107Thr + Asn162Glu + Asp165Glu +
Thr217ProVal 95Glu + Ser101Asp + Asn162Glu + Phe192Thr +
Gly215GlnGln103Asn + Tyr104His + Thr206Glu + Ser216Glu +
Thr223GluGly135Asp + Asn168Asp + Tyr169Asn + Ser191Glu +
Gly205ProGly102Glu + Ser105Glu + Asn161Gln + Ser207Glu +
Ile220MetLeu133Glu + Phe192Pro + Thr206Glu + Thr217Pro +
Ser219GluAsn 99Asp + Ser105Glu + Leu133Val + Ser140Glu +
Gly214SerAsn 99Glu + Gly102Asp + Asn162Gln + Phe192Pro +
Ser219AspIle107Val + Ser190Glu + Asn194Glu + Leu209Thr +
Ser216GluGly100Glu + Leu133Glu + Ala166Thr + Ser216Asp +
Thr217ProArg 64Asp + Ala164Gln + Ser191Asp + Thr206Glu +
Leu209CysLeu 96Val + Gly134Asp + Gly136Pro + Ser191Asp +
Thr206GluTyr104Asn + Asp165Glu + Phe192Asp + Ser207Asp +
Ile220GlnSer101Asp + Ala164Ser + Thr206Pro + Ser207Glu +
Arg218AspAsp165Glu + Ser207Glu + Leu209Asn + Trp212Gly +
Arg218AspGly100Asn + Tyr104Pro + Ser138Glu + Ser207Glu +
Arg218GluSer140Glu + Ser170Glu + Thr211Gln + Gly215Asn +
Ser221AspGly100Glu + Ser140Asp + Ser170Asp + Thr206Ser +
Leu209HisSer140Glu + Tyr169Pro + Ser170Glu + Arg218Asp +
Ile220ProGln103Ser + Tyr104Asp + Leu209Glu + Ser221Asp +
Gly222SerAsp 97Glu + Ile107Cys + Asn162Asp + Ser191Asp +
Leu209AlaAsp 97Glu + Tyr104Glu + Ile107Gly + Leu133Ser +
Ile220LeuLeu 96Glu + Leu133Asp + Gly134Asn + Gly215Ser +
Ser216GluAsn 67Glu + Leu 96Glu + Thr106Pro + Ile107Cys +
Ser207GluArg 64Glu + Asp 97Glu + Gly102Asn + Asp165Glu +
Phe192TyrGly 66Ser + Gly134Asp + Asn161Asp + Ser207Glu +
Thr217GlyAsp 65Glu + Gly 70Asn + Gly100Pro + Leu209Asp +
Ser216AspGly 68Glu + Ser101Asp + Leu209Thr + Trp212Ala +
Ser219AspThr106Gly + Asn163Asp + Leu209Pro + Ser219Glu +
Thr223GluAsp 65Glu + Gln103Ser + Gly134Glu + Gly136Asn +
Ser138GluGln103Ser + Ile107His + Asn161Asp + Asp165Glu +
Ser207GluGly 66Pro + Ser140Glu + Ala164His + Thr217Asp +
Ile220AspArg 64Asp + Ser101Asp + Tyr104Leu + Ile107Glu +
Ile220AsnArg 64Glu + Val 95Met + Gly136Ser + Ser190Glu +
Ile213AspGly135Glu + Gly160Pro + Leu209Gln + Ile220Leu +
Thr223GluAsp 65Glu + Val 95Gln + Ser170Glu + Phe192Met +
Gly214GluGly100Asn + Ser190Asp + Ser207Asp + Leu209Ser +
Ser216GluVal 95Gly + Arg167Asp + Ser190Asp + Ser207Glu +
Ile220AsnAsn 67Glu + Ser101Glu + Leu133Ser + Asn163Asp +
Thr223SerAsn 67Glu + Asn 99Ser + Ser101Glu + Tyr104Ala +
Ser207GluGly134Asn + Ser138Asp + Arg218Asp + Ile220His +
Ser221GluGly 66Gln + Val 95Pro + Ser138Glu + Arg218Glu +
Ser221AspAsn 67Asp + Thr 71Ser + Gly135Glu + Asn161Asp +
Ala164HisAsp 65Glu + Gly 66Pro + Ser191Glu + Ile213Ser +
Ser221GluGly 70Pro + Gly134Pro + Ser191Asp + Ser216Asp +
Ser221AspGly100Glu + Tyr104Glu + Asn163Gln + Ile213Pro +
Ile220SerGly134Glu + Gly136Pro + Asn162Gln + Ser216Asp +
Thr223AspThr 71Asn + Ser105Asp + Leu209Met + Ser219Asp +
Gly222GluGly 66Asn + Gly102Asn + Tyr169Asp + Ser216Asp +
Ser219AspGly136Pro + Asn161Glu + Ser216Glu + Ser219Glu +
Ile220SerSer140Glu + Ile208Met + Ser216Glu + Ser219Glu +
Ile220AsnGly160Asp + Thr206Gln + Gly214Ser + Ser216Glu +
Ser219GluAsn 99Ser + Ser190Glu + Ser216Glu + Ser219Glu +
Ile220HisSer139Asp + Thr206Gln + Ser216Glu + Ser219Asp +
Gly222AsnGly102Asn + Ser190Asp + Ser216Glu + Ser219Asp +
Gly222GlnGln103Ser + Asn163Asp + Trp212Met + Ser216Asp +
Ser219AspThr 71Gly + Gly136Glu + Asn194Gln + Ser216Asp +
Ser219GluAsp 97Glu + Leu209Met + Ser216Glu + Ser219Asp +
Ile220ValAsn 99Gln + Asp165Glu + Leu209Met + Ser216Glu +
Ser219GluThr106Glu + Asn168Gln +Leu209Met + Ser216Asp +
Ser219GluLeu 96Gln + Asn161Gln + Tyr169Asp + Ser216Asp +
Ser219GluLeu 96Met + Asn161Asp + Gly203Gln + Ser216Asp +
Ser219GluAsn 99Glu + Thr206Ser + Ser207Glu + Leu209Val +
Thr223GluSer207Glu + Leu209Met + Ser216Asp + Ile220Leu +
Thr223AspVal 95Ala + Gly214Asn + Ser216Asp + Ile220Asp +
Gly222ProSer101Asp + Tyr104Gln + Gly136Asn + Gly160Glu +
Ser190AspGly 70Asp + Gly135Glu + Arg167Asp + Asn168Gln +
Ile220GlnAsp 65Glu + Leu 96Met + Gly136Glu + Asn162Asp +
Leu209AlaVal 95Asp + Asn 99Glu + Asn194Ser + Gly214Pro +
Thr223AspGly136Glu + Ala164Ser + Asp165Glu + Tyr169Pro +
Ser216AspArg 64Glu + Gly135Asn + Asn162Gln + Ser19lAsp +
Gly214AspArg 64Asp + Asn162Gln + Thr206Gln + Ser207Glu +
Gly214GluLeu 96Ala + Gln103Ser + Asn162Glu + Arg167Asp +
Gly215GluAsn161Ser + Asn162Glu + Arg167Glu + Thr206Pro +
Ser219AspAla164His + Asp165Glu + Tyr169Pro + Ser170Asp +
Gly215GluVal 95Ala + Gln103Glu + Ser138Asp + Gly160Asn +
Ser219AspIle107Glu + Leu133Asn + Ser138Asp + Ala164Gly +
Ser191GluIle107Glu + Leu133Ser + Asn163Glu + Arg167Glu +
Leu209HisSer101Glu + Leu209Asp + Gly215Gln + Ile220Asn +
Gly222GluSer139Asp + Asn162Gln + Tyr169Gln + Leu209Asp +
Gly222AspThr106Glu + Gly136Ser + Ser170Glu + Asn194Glu +
Thr206GlnTyr104Ala + Leu133Asn + Asn162Asp + Ser207Asp +
Thr217AspTyr169Cys + Ser191G1u + Ser207Glu + Ser216Asp +
Gly222GlnVal 95Gln + Tyr104Leu + Tyr137Glu + Ser191Glu +
Ser207GluGly 66Asp + Gly100Asn + Ser101Glu + Ser139Glu +
Tyr169GlnGly 66Glu + Val 95Ser + Ser101Glu + Asn163Ser +
Ser191AspArg 64Glu + Val 95Ala + Asn 99Glu + Ala164Glu +
Gly214SerArg 64Glu + Asn 99Asp + Ser105Asp + Gly160Pro +
Leu209GlyAsn 67Asp + Val 95Asp + Gly102Pro + Ser207Glu +
Gly214SerSer101Asp + Ser105Asp + Asn162Glu + Tyr169Gln +
Ile220SerSer139Asp + Gly160ser + Ala166Thr + Phe192Glu +
Ser207AspVal 95Gly + Gly136Pro + Asn161Glu + Arg167Asp +
Ser207AspAsp 65Glu + Ser101Glu + Gly102Asn + Leu133Thr +
Ser170AspGly136Gln + Asn161Asp + Arg167Asp + Asn194Ser +
Ser216GluGly135Asp + Ala164Asp + Ser216Glu + Ile220Leu +
Gly222AsnAsp 97Glu + Asn 99Gln + Gln103Asn + Ser105Glu +
Ala166SerAsn 99Gln + Asn163Glu + Ile213Leu + Ile220His +
Ser221AspGly135Glu + Asp165Glu + Phe192Gln + Ser207Glu +
Gly215SerAsn162Asp + Ser170Glu + Asn194Ser + Leu209Gln +
Ser219GluGly100Asp + Thr106Asp + Ala164Glu + Phe192Thr +
Asn194GlnSer105Glu + Asn162Glu + Leu209His + Ser221Glu +
Gly222GlnGly 68Asn + Gly135Glu + Gly160Gln + Ser216Glu +
Ile220AspAsp 97Glu + Ala164Gly + Leu209Met + Ser216Asp +
Ile220GluGly 68Asp + Tyr137Asp + Asn194Gln + Leu209Cys +
Ser221AspIle107Asp + Gly136Gln + Ser139Asp + Ser216Asp +
Ile220ThrGly100Pro + Ser101Glu + Gly136Glu + Ser191Asp +
Phe192AlaGly 70Glu + Gly160Pro + Ala164Thr + Ser170Glu +
Ser216GluArg 64Asp + Tyr104His + Asp165Glu + Leu209Gln +
Thr223GluLeu133Ala + Ser138Asp + Leu209Cys + Thr217Glu +
Ser221AspSer140Glu + Gly203Asn + Thr217Glu + Ile220Met +
Ser221AspAsp 97Glu + Thr106Asn + Gly134Glu + Asp165Glu +
Thr211GlnGly102Pro + Ser140Asp + Asn194Asp + Gly215Pro +
Ser221AspArg167Asp + Leu209Asn + Ser219Asp + Ile220Val +
Thr223GluGly 68Asp + Gly102Asp + Gly136Ser + Ser170Asp +
Phe192Leu
表36
多环区六突变变异体Val 95His + Ala164Ser + Ser170Glu + Phe192Met +
Leu209Thr + Ile220ProLeu133Asn + Asn161Gln + Asn163Gln + Leu209Met +
Ile220Leu + Thr223GlyGly134Asn + Tyr169Ala + Thr211Pro + Ser216Asp +
Ile220Met + Gly222GlnGly136Glu + Gly203Gln + Thr206Pro + Leu209Thr +
Gly215Pro + Ile220MetGly100Ser + Gly135Asn + Ala164Thr + Gly214Glu +
Thr217Gln + Ile220LeuThr106Ser + Thr206Pro + Ile208Cys + Leu209Ser +
Ser216Asp + Ile220SerGly 68Asn + Leu 96His + Gly102Asn + Leu209Cys +
Trp212Ala + Ile220ValGly 70Pro + Val 95Gln + Gly102Gln + Asn162Asp +
Gly214Gln + Ile220HisAsn 67Gln + Leu 96Val + Leu133Ala + Ala164Thr +
Leu209Ala + Ser219AspGly100Asn + Leu133Asp + Asnl61Gln + Thr206Gly +
Ile213Leu + Gly222GlnLeu 96Asn + Gly100Gln + Thr106Asn + Ser140Glu +
Tyr169His + Ile220AsnGly 70Pro + Asn 99Ser + Gly135Gln + Tyr169Gly +
Thr206Ser + Ser216AspSer101Glu + Gly102Glu + Tyr169Met + Thr206Asn +
Leu209Ile + Ile220GlnVal 95Ala + Asn168Gln + Gly214Asp + Gly215Glu +
Ser216Glu + Ile220ProLeu 96Pro + Thr106Gly + Ile107Gln + Asn163Glu +
Phe192Glu + Leu209GlyAsn194Ser + Phe202Gly + Ser207Glu + Arg218Glu +
Ser219Asp + Ile220AspAsp 65Glu + Asp 97Glu + Asp 98Glu + Asn168Ser +
Tyr169Thr + Thr211SerAsn 67Gln + Gly215Ser + Ser219Glu + Ile220Pro +
Ser221Glu + Gly222GlnGly102Asn + Gln103Ser + Tyr169Ile + Thr206Gly +
Ser216Glu + Arg218AspAsn 67Glu + Gly 70Glu + Leu 96Pro + Asn 99Ser +
Asn162Gln + Phe192ProAsn 67Asp + Gly 68Gln + Gly 70Asp + Leu133Val +
Leu209Gly + Thr217AsnGly 68Ser + Leu133Glu + Gly135Asp + Ser170Glu +
Ile213Asn + Ile220SerVal 95Asp + Asp 97Glu + Gly100Asp + Gly135Gln +
Leu209Gln + Thr223SerLeu 96Cys + Gly134Asp + Gly160Glu + Asn161Glu +
Asn162Glu + Leu209ProAsp 65Glu + Leu 96Glu + Asp 97Glu + Asn 99Asp +
Ile220Ser + Gly222ProVal 95Pro + Leu209Ser + Ser219Glu + Ile220Gly +
Ser221Glu + Gly222AspGly 66Asn + Asp 98Glu + Ser101Asp + Tyr104Cys +
Asn194Gln + Ile220AsnIle107Thr + Tyr137His + Phe202Leu + Ser216Glu +
Arg216Asp + Ser219GluGly134Pro + Tyr137Pro + Ser207Glu + Leu209Cys +
Ser219Asp + Gly222GluLeu133Thr + Asn163Ser + Ser207Asp + Ile213Met +
Arg218Asp + Ser221AspArg 64Asp + Asp 98Glu + Thr106Ser + Gly160Asn +
Ile213Pro + Ile220AsnGly 70Gln + Leu 96Asn + Tyr169Met + Thr206Glu +
Leu209Cys + Thr223AspGly 68Pro + Ala164Glu + Arg167Glu + Pro204Asn +
Thr206Gln + Ile220SerAsn 99Ser + Gly135Asp + Tyr137Met + Ser138Glu +
Asn168Asp + Thr211AsnGly 68Pro + Tyr104Glu + Ser138Glu + Thr217Ser +
Ile220Gln + Thr223ProGly 66Ser + Asn163Gln + Thr206Asp + Leu209Gly +
Ser219Asp + Ile220MetGly 68Asp + Thr106Pro + Thr206Gly + Leu209Glu +
Ser219Glu + Ile220AspTyr137Leu + Tyr169Cys + Phe192Asn + Ser207Asp +
Arg218Glu + Ile220LeuGly 68Asn + Val 95Glu + Gly100Glu + Leu133Cys +
Gly134Pro + Thr217SerGly 68Asp + Gly100Gln + Thr206Pro + Ser219Glu +
Ile220Glu + Ser221GluTyr137Asn + Ala164Gln + Ser207Glu + Leu209Glu +
Arg218Asp + Gly222GluGly135Pro + Ser191Asp + Asn194Gln + Thr206Glu +
Ser207G1u + Ser219GluVal 95Asp + Ser105Asp + Gly160Asn + Leu209Gly +
Gly215Pro + Ile220AlaGly 68Asp + Gly100Ser + Gly134Asn + Ser207Glu +
Ser219Glu + Ser221GluAsn 67Gln + Gln103Ser + Thr206Glu + Leu209Glu +
Ser216Glu + Ser219AspTyr104Ile + Thr206Glu + Ser216Asp + Arg218Asp +
Ser219Glu + Thr223SerGly 68Gln + Val 95Pro + Leu133Ala + Asn162Gln +
Ser190Glu + Ser207AspAsn163Ser + Thr206Asp + Ser207Glu + Leu209Ser +
Ser216Glu + Ser219AspAsn161Ser + Thr206Ser + Gly214Asp + Gly215Asp +
Thr217Glu + Ile220GluAsn 99Ser + Ser138Asp + Ser139Asp + Ser140Asp +
Ile213Gly + Ser216AspGly 70Glu + Leu 96Asn + Thr206Asn + Ser207Glu +
Ser219Asp + Ser221AspTyr104His + Asn161Asp + Ser170Asp + Phe192His +
Asn194Asp + Gly222ProLeu 96Glu + Asp 97Glu + Ser101Asp + Leu209Thr +
Ser219Asp + Ile220AsnSer207Asp + Ile208Ala + Leu209Thr + Ser216Asp +
Ser219Glu + Ile220LeuGly100Glu + Leu133Glu + Gly135Glu + Asn161Glu +
Ala164Gly + Leu209ThrLeu 96Asn + Gly136Ser + Leu209His + Gly214Glu +
Ser216Asp + Ser219GluTyr104Leu + Ile107Glu + Tyr137Glu + Ser138Asp +
Asn168Gln + Leu209IleTyr137Glu + Phe192Met + Ser207Glu + Ser219Asp +
Ser221Glu + Thr223ProTyr137Asp + Asn168Asp + Ser170Glu + Phe192Asn +
Ser207Asp + Ile220AsnVal 95Ser + Gly102Ser + Asn162Gln + Leu209Ser +
Ser216Glu + Ser219AspGly 66Ser + Gly102Gln + Ser216Glu + Ser219Asp +
Ile220Val + Gly222AsnAsp 97Glu + Asp 98Glu + Ser101Glu + Gly134Pro +
Asn162Glu + Thr206GlnVal 95Pro + Ser101Asp + Gly135Asn + Ser207Asp +
Leu209Asp + Ile220AspAsp 98Glu + Gly203Ser + Ser207Glu + Leu209Pro +
Arg218Glu + Ser219AspThr106Pro + Gly135Asp + Ser140Asp + Asn162Glu +
Ser170Glu + Gly214GlnGly 68Gln + Ile107Asn + Asn162Ser + Ser191Asp +
Ser207Asp + Ser219GluGly102Ser + Gly136Asn + Thr206Glu + Leu209Thr +
Arg218Asp + Gly222AspAsp 65Glu + Gln103Ser + Ser207Glu + Leu209Gln +
Arg218Asp + Ile220GluGly 66Pro + Asn 99Gln + Gly136Asp + Arg218Asp +
Ile220Glu + Ser221AspTyr104Asp + Gly134Glu + Gly135Asp + Thr211Ser +
Ser219Glu + Ile220ValGly100Gln + Tyr104Ser + Ser139Glu + Ser191Glu +
Phe192Asp + Thr206GluSer138Glu + Thr206Pro + Leu209Thr + Ser216Glu +
Arg218Asp + Ser219AspAsp 97Glu + Leu209Gln + Ser216Asp + Arg218Asp +
Ser219Glu + Ile220AlaGly102Asp + Leu133Thr + Asn163Gln + Ser216Glu +
Thr217Glu + Ser219AspAsp 98Glu + Asn 99Asp + Gly102Asp + Thr106Ser +
Ile107Gln + Ser138AspAsn 67Asp + Gly 70Glu + Ser170Asp + Phe192Pro +
Leu209Thr + Ile213GluGln103Asp + Gly134Asn + Asn162Glu + Phe192Glu +
Ile220Leu + Gly222AspAsp 65Glu + Asp 97Glu + Gly100Asp + Tyr169Met +
Ser191Glu + Ile220CysAsn 67Asp + Asp 98Glu + Ala164Pro + Ile208Thr +
Leu209Ser + Ile220AspGly136Asn + Ser191Asp + Thr206Pro + Arg218Asp +
Ser219Asp + Gly222AspAsp 98Glu + Gly136Asp + Ser138Glu + Ser140Asp +
Gly203Gln + Ile220MetLeu133Ile + Gly134Pro + Gly135Pro + Asn162Glu +
Arg167Glu + Ile213ValGly102Asn + Ser140Asp + Asn161Glu + Asn162Asp +
Ser190Glu + Gly205AsnSer139Glu + Gly160Glu + Asn161Gln + Thr206Pro +
Ser221Glu + Gly222GluGly 66Asp + Asn 67Asp + Ile107Gly + Asn194Gln +
Ser207Asp + Ile220GluTyr104Gly + Gly136Asp + Ser170Asp + Ser190Glu +
Asn194Asp + Gly222SerGly 68Asp + Ile107Asp + Gly135Asn + Gly215Glu +
Ser216Glu + Ile220GlySer101Glu + Ser207Asp + Leu209Met + Gly215Pro +
Thr217Glu + Ser219GluAsp 65Glu + Gly100Glu + Gln103Asp + Asn161Gln +
Phe192His + Ile220SerGly 68Gln + Ser138Asp + Ser139Glu + Ala164Gly +
Asn194Glu + Phe202GlnAsn 99Glu + Thr106Gln + Gly214Gln + Gly215Asn +
Ser219Asp + Ile220GluGly 66Pro + Asn163Glu + Ala164Gln + Phe192His +
Ser219Asp + Ile220AspGly100Gln + Tyr104Pro + Tyr137His + Arg167Asp +
Asn168Asp + Ser207AspAsn 99Ser + Gly100Ser + Asn161Asp + Asn162Asp +
Leu209Asn + Ser216GluGly100Asp + Ser101Glu + Asn168Gln + Leu209Met +
Ile213Ser + Ser219AspVal 95His + Gly100Gln + Ser139Glu + Ser140Asp +
Ser207Glu + Thr217SerGly100Asp + Gly134Gln + Ser139Asp + Ser140Asp +
Asn161Ser + Leu209ValVal 95Cys + Gly100Glu + Thr106Asn + Ser139Asp +
Ser140Glu + Gly222SerSer105Asp + Asn194Gln + Thr206Asp + Ser207Asp +
Leu209Val + Gly215SerAsn163Glu + Phe192Pro + Trp212Val + Ser216Asp +
Thr217Asp + Gly222ProVal 95Asn + Gln103Asn + Leu209Gln + Gly215Asp +
Ser216Glu + Ser221AspGly 66Gln + Gly134Glu + Ser170Asp + Leu209Thr +
Ile220Asp + Gly222AspGly 66Gln + Thr106Glu + Ile107Glu + Asn163Asp +
Asn194Glu + Pro204AsnGly102Glu + Gln103Asp + Phe192Ile + Ser207Glu +
Leu209Pro + Ile220AspArg 64Glu + Asp 65Glu + Ala164Asn + Ser207Glu +
Leu209His+ Ser219AspArg 64Asp + Asp 65Glu + Gly135Pro + Tyr137Met +
Ser207Glu + Ser219AspTyr104Gly + Ser105Glu + Phe192Gln + Ser207Asp +
Ser221Asp + Gly222GlnSer105Glu + Tyr137Ile + Ala164His + Thr206Asn +
Ser207Glu + Ser221GluAsn 67Gln + Asp 98Glu + Asn161Gln + Asn168Ser +
Ser207Glu + Ile220AspGly134Ser + Ser139Asp + Thr206Asp + Ile213Ala +
Arg218Glu + Ser219AspAsp 98Glu + Gly100Gln + Gln103Asn + Gly134Asp +
Ser139Glu + Ser170GluLeu 96Gly + Asp 98Glu + Thr106Asn + Leu209Glu +
Ser216Asp + Ser219AspGly 70Glu + Asp 97Glu + Asn 99Glu + Gly135Ser +
Asn194Glu + Leu209ThrThr106Gly + Arg167Glu + Ser207Glu + Gly214Gln +
Gly215Pro + Ser219GluAla164Glu + Thr206Gly + Ser207Asp + Leu209His +
Ser219Asp + Ile220GlnGly160Ser + Asn162Gln + Ser170Glu + Thr206Pro +
Ser207Asp + Ser219AspTyr104Cys + Asn162Gln + Tyr169Asp + Ser207Glu +
Leu209Ala + Ser219AspAsn 67Gln + Asp165Glu + Ser207Glu + Leu209Val +
Ser219Asp + Ile220AlaGly 66Glu + Thr106Gln + Phe192Asp + Asn194Ser +
Thr206Glu + Ser207AspGly134Asp + Ala164Ser + Ser170Asp + Ser207Asp +
Ile208Met + Ser219AspGly 66Gln + Gly102Asn + Gly136Asp + Ser139Glu +
Asn168Asp + Ser219AspAsp 97Glu + Asn 99Asp + Thr206Asn + Ser207Asp +
Ser219Asp + Ile220MetIle107Val + Asn162Asp + Ser191Asp + Ser207Asp +
Ser219Glu + Gly222ProGly 66Ser + Asn 67Glu + Ser138Asp + Ser140Glu +
Leu209Cys + Ile213AlaGly 70Ser + Asp 98Glu + Gly100Asp + Asn162Gln +
Ser207Glu + Ser219GluAsp 97Glu + Asn 99Glu + Tyr104Gly + Thr206Glu +
Leu209Ala + Thr217GlnArg 64Glu + Gly 66Glu + Asn162Asp + Tyr169Ser +
Leu209His + Gly214GluGly 70Gln + Leu133Asp + Gly136Glu + Asn194Asp +
Ile213Cys + Gly222AspGly 70Gln + Gln103Asp + Ser105Glu + Thr106Pro +
Ser219Asp + Ile220SerGln103Glu + Ser105Glu + Asn162Gln + Gly205Pro +
Trp212Asn + Arg218GluAsp 97Glu + Asn 99Gln + Gly136Asp + Gly160Ser +
Ser170Asp + Leu209MetSer105Glu + Ile107Glu + Ser207Glu + Ile213Thr +
Gly215Ser + Ile220AsnSer190Asp + Phe192Ile + Ser216Glu + Ser219Glu +
Ile220Glu + Gly222SerAsp 65Glu + Gly102Gln + Ser207Asp + Ile213Asp +
Ser216Glu + Ile220AsnGln103Asp + Leu133Asn + Phe192Asp + Ile213Val +
Ile220Asp + Ser221AspLeu 96Cys + Ile107Gly + Asn162Asp + Asn194Asp +
Ser216Asp + Ile220HisSer101Asp + Ser105Glu + Thr106Asn + Ser138Asp +
Ser170Glu + Leu209SerGly 70Ser + Tyr169Val + Phe192Asp + Ser216Glu +
Ser2l9Glu + Ile220AspGly136Gln + Asn161Asp + Asn163Gln + Arg167Asp +
Asn194Glu + Ser216GluAsp 97Glu + Asp 98Glu + Asn161Glu + Ala164Asp +
Ile213Ala + Thr217GlyIle107Cys + Arg167Asp + Asn168Glu + Ser207Glu +
Ile220Thr + Gly222GluArg167Asp + Tyr169His + Ser170Glu + Leu209Val +
Ser219Asp + Ile220GluGly 70Gln + Phe192Asp + Ser216Glu + Arg218Glu +
Ile220Val + Gly222GluArg 64Asp + Gly 70Asn + Phe192Ala + Ser207Asp +
Leu209Glu + Thr223GlyGly 68Asn + Asp 98Glu + Ser207Asp + Leu209Asp +
Ile220Asn + Thr223GlnGly100Gln + Leu133Ile + Asp165Glu + Ala166His +
Tyr169Asp + Ser191GluAsn163Asp + Ser191Asp + Thr206Gly + Leu209Asp +
Thr211Asn + Ile220GluAsn 99Gln + Gly160Glu + Tyr169Val + Ser191Asp +
Ser216Asp + Gly222AspAsn 67Glu + Asp 97Glu + Ser105Asp + Ala164Thr +
Thr206Ser + Ile220ValArg 64Asp + Gly 70Glu + Val 95Thr + Asp 97Glu +
Asn162Asp + Thr223GlnLeu 96Ser + Gln103Glu + Ser105Glu + Gly134Asp +
Asn194Ser + Ser219AspGly100Gln + Leu133Gln + Ala164Gln + Arg167Glu +
Ser219Asp + Ser221GluVal 95Gln + Tyr137Asn + Ser140Glu + Pro204Asn +
Ser219Asp + Ser221AspGly134Ser + Ser190Glu + Ser207Glu + Leu209Gln +
Thr217Glu + Ile220HisGly 70Ser + Gly134Ser + Asn162Ser + Ser170Asp +
Ser216Glu + Arg218AspAsp 97Glu + Tyr137Thr + Tyr169Gln + Ser216Glu +
Arg218Glu + Gly222GlnAsn 67Glu + Gly 70Glu + Asn 99Gln + Tyr104Met +
Gly136Asp + Ala164AsnAsn 67Glu + Gly 68Asp + Gly 70Gln + Ser191Glu +
Ile213Ser + Ile220GluThr106Gly + Asn162Glu + Gly215Glu + Thr217Asp +
Ser219Glu + Ile220AsnArg 64Asp + Asp 65Glu + Ile208Gln + Leu209Asp +
Ser216Asp + Ile220ValSer101Glu + Leu133Glu + Thr206Asn + Ser207Glu +
Thr217Asn + Ser219AspTyr104Glu + Ser138Glu + Asn162Glu + Asn194Glu +
Thr211Ser + Trp212SerAsp 98Glu + Asn 99Gln + Gly102Glu + Ser190Glu +
Ser191Glu + Ile220ProVal 95Asp + Gly134Gln + Tyr169Pro + Phe192Asn +
Ser221Glu + Thr223AspAsn 67Glu + Leu 96Glu + Gly160Ser + Asn162Ser +
Thr206Gly + Thr217AspGly 68Asp + Val 95Glu + Asn 99Glu + Tyr169Ala +
Gly215Asp + Thr217SerVal 95Ser + Gly135Ser + Asn161Asp + Ile213Asp +
Ser216Asp + Ser219AspArg 64Glu + Asp 98Glu + Gly136Ser + Gly160Gln +
Leu209Asp + Ile220AspAsp 65Glu + Gly 66Ser + Asn194Gln + Ser207Asp +
Ser216Asp + Ser219AspAsn 99Ser + Ser101Asp + Tyr104Asp + Gly134Ser +
Ser207Asp + Ile220AspArg 64Glu + Asp 98Glu + Ile107Asn + Thr206Ser +
Ser219Asp + Ser221GluVal 95Pro + Ala164Asp + Asp165Glu + Ser170Asp +
Ile213Asp + Ile220ValGly135Ser + Asn161Gln + Asn162Asp + Tyr169Asp +
Gly215Gln + Ser221GluTyr137His + Ser140Asp + Asn161Gln + Ser170Asp +
Ser207Asp + Leu209AspLeu 96Ile + Ser138Glu + Ala164His + Asn194Asp +
Thr206Asp + Gly222GluAsn161Glu + Ser207Asp + Gly214Asn + Ser216Asp +
Arg218Glu + Gly222GlnLeu 96Asn + Ser170Asp + Ile208Asn + Gly215Glu +
Arg218Glu + Ser219AspGly134Asp + Ser139Asp + Ser140Glu + Thr206Asp +
Ile213Met + Thr223AsnAsp 65Glu + Asp165Glu + Phe192Pro + Leu209Gly +
Gly215Glu + Ser216AspAsn 99Asp + Ser101Glu + Ile107Glu + Ser191Glu +
Asn194Gln + Ile220LeuAsp 65Glu + Ser190Glu + Asn194Gln + Ser207Glu +
Gly215Pro + Ser219GluGly134Asn + Ser138Glu + Ser139Glu + Ser216Glu +
Ser219Asp + Ile220ProTyr137Cys + Ala164Thr + Ser190Glu + Ile208His +
Ser216Glu + Gly222GluAsp165Glu + Ser207Asp + Ser216Glu + Arg218Asp +
Ile220Pro + Gly222ProVal 95Met + Ser139Glu + Ser140Asp + Ala164Asn +
Ser216Asp + Ser219AspVal 95Gly + Gly102Ser + Ser105Asp + Ser138Glu +
Asn163Glu + Ala164GluAsp 65Glu + Val 95Gly + Asp 97Glu + Gly160Ser +
Asp165Glu + Ile220SerThr 71Gln + Ser101Asp + Thr106Glu + Tyr169Pro +
Ser207Glu + Ser219AspTyr137Ile + Asp165Glu + Tyr169Cys + Gly214Asp +
Arg218Glu + Ser219AspAsn 99Ser + Ala164Asp + Ser170Asp + Asn194Glu +
Thr206Gln + Ser216AspAsp 65Glu + Gly134Asp + Gly160Asn + Asn161Glu +
Asn168Asp + Ile220AsnAsp 65Glu + Phe192Ser + Ser216Glu + Ser219Glu +
Ile220Met + Ser221GluGly 68Asn + Leu133Gln + Asn168Asp + Ser207Glu +
Ser216Glu + Ser219AspGly135Glu + Ser207Glu + Ser216Asp + Thr217Asn +
Ser219Glu + Ile220ProGly 68Asn + Asn163Glu + Ser207Asp + Ser216Glu +
Ser219Glu + Ile220AsnGly102Glu + Tyr137Cys + Thr206Ser + Ser207Glu +
Ser216Asp + Ser219AspLeu133Ser + Arg167Glu + Ser170Asp + Ser191Asp +
Thr206Asp + Leu209HisGly 66Gln + Gly136Glu + Ser138Glu + Thr206Gln +
Arg218Glu + Ser221AspAsn 67Glu + Ser139Asp + Ile208Val + Leu209Ser +
Ser216Asp + Thr217AspArg 64Asp + Val 95His + Gly134Glu + Ser138Glu +
Arg167Glu + Leu209CysArg 64Asp + Phe192Val + Thr206Asp + Leu209Ala +
Ser219Glu + Gly222SerLeu 96Ala + Ser140Asp + Phe202Gln + Thr206Asp +
Leu209Ala + Ser219GluLeu 96Ile + Gly102Pro + Gly134Asp + Thr206Asp +
Leu209Ala + Ser219AspGly102Glu + Ser105Asp + Ile107Cys + Ser140Asp +
Asn194Asp + Ile220AlaGly100Glu + Ser105Asp + Tyr137Pro + Ser140Glu +
Gly203Asn + Gly222ProThr106Asn + Asn162Glu + Asn168Asp + Ser216Glu +
Arg218Asp + Ile220AsnAsp 98Glu + Leu133Gly + Gly136Asp + Ser140Glu +
Asn168Glu + Phe192SerVal 95Ala + Asp165Glu + Asn168Ser + Ser207Asp +
Leu209Glu + Ser216AspGly134Glu + Ala164Asp + Asn168Glu + Phe192Cys +
Ser219Glu + Ile220ProGly 68Asn + Ser191Asp + Phe192Asn + Gly215Glu +
Ser219Glu + Ser221GluGln103Glu + Ile107Asn + Ser140Glu + Ser190Glu +
Ser191Glu + Leu209ThrTyr104Ala + Leu133Asn + Asn162Asp + Thr206Glu +
Ser207Asp + Thr217AspGly160Asn + Asn194Asp + Ser207Asp + Ile213Val +
Ser216Asp + Thr217AspLeu 96Ile + Ala164Gln + Asp165Glu + Ser207Glu +
Arg218Glu + Ile220MetGly 66Glu + Ile107Ala + Gly160Asn + Phe192Val +
Ser207Glu + Arg218GluGly100Gln + Tyr104Asp + Gly134Asp + Ala164Ser +
Phe192Leu + Ser216AspArg167Glu + Leu209Thr + Ser216Glu + Ser219Glu +
Ser221Glu + Gly222AsnGly 68Asn + Asp 98Glu + Leu209Gly + Ser216Asp +
Ser219Asp + Ser221GluAsp 65Glu + Ser207Glu + Leu209Asp + Gly215Asp +
Gly222Ser + Thr223AsnGly134Ser + Ser140Glu + Asn168Gln + Ser191Glu +
Asn194Glu + Ser221AspLeu 96Ile + Thr106Glu + Gly160Asp + Ser219Glu +
Gly222Glu + Thr223SerSer101Asp + Ser105Glu + Asn161Gln + Ser207Glu +
Ile213Asn + Ser221GluGln103Ser + Gly135Asp + Ser190Glu + Thr206Ser +
Ser219Glu + Ile220GluAsp 65Glu + Gly100Asp + Ser105Asp + Ser140Glu +
Phe192Thr + Ile220HisAsp 65Glu + Ser101Glu + Ala164Thr + Ile213Glu +
Thr217Asn + Ser219GluSer105Asp + Gly134Asp + Asn168Ser + Ser191Asp +
Phe192Glu + Ile220CysLeu 96Asp + Ile107Asp + Gly214Ser + Ser216Glu +
Ser219Glu + Ile220ValAsn162Gln + Asp165Glu + Arg218Asp + Ser219Glu +
Ile220Thr + Thr223AspThr 71Gly + Asn161Asp + Asn168Asp + Ser207Glu +
Leu209Asp + Thr223GlnAsp 65Glu + Asp 98Glu + Leu133Ser + Asp165Glu +
Leu209Met + Gly215GluArg 64Asp + Asn 99Glu + Asp165Glu + Ala166Asn +
Asn194Glu + Ile220CysAsn 99Ser + Gly136Asp + Asn163Asp + Asp165Glu +
Phe202Cys + Ser216GluVal 95Pro + Leu133Thr + Gly136Glu + Asp165Glu +
Ser216Asp + Arg218GluLeu133Pro + Gly134Asn + Asn194Asp + Thr206Glu +
Gly214Glu + Ser221AspGly 70Asp + Asp 97Glu + Tyr104Asn + Pro204Gly +
Leu209Asp + Ser216GluGln103Asp + Gly135Asp + Ser140Asp + Asn168Gln +
Gly214Asp + Ile220LeuAsp 65Glu + Thr 71Ser + Asn 99Glu + Ser138Asp +
Phe192Tyr + Leu209GlyTyr104Val + Ile107Glu + Thr206Asp + Ser216Asp +
Ser219Glu + Ile220LeuLeu133Ala + Arg167Glu + Leu209Gln + Ser216Glu +
Thr217Glu + Ser221GluAsn 67Glu + Asp 98Glu + Tyr137Ala + Asp165Glu +
Thr206Pro + Ile220AspSer101Glu + Ile107Asp + Asn161Glu + Asp165Glu +
Ile220Thr + Thr223GlnTyr104Asp + Thr106Asn + Tyr137Glu + Gly160Gln +
Asn162Asp + Gly215SerAsn 67Asp + Asp 97Glu + Tyr104Thr + Leu133Asp +
Ala164Glu + Ile220AlaAsp 97Glu + Asn 99Ser + Ser105Asp + Thr206Asp +
Ser207Glu + Trp212ThrAsn 67Gln + Tyr104Ile + Ser105Glu + Asn163Glu +
Ile220Glu + Ser221GluAsp 98Glu + Asn 99Gln + Leu133Asp + Phe192Glu +
Asn194Glu + Gly205ProAsn 99Asp + Ile107Ser + Tyr137Met + Asn161Glu +
Asp165Glu + Ile213AsnGly 70Asn + Gln103Ser + Ser138Glu + Ala164Asp +
Ser170Glu + Ser219AspSer101Glu + Thr106Asn + Gly135Glu + Leu209Ser +
Thr217Asn + Ser219GluGly 68Glu + Gly102Gln + Gly135Ser + Ser190Asp +
Ser207Asp + Ile220AlaAsp 65Glu + Val 95Glu + Gly100Gln + Ser105Asp +
Asn194Ser + Leu209GluAsp 65Glu + Tyr137His + Asn161Glu + Asn162Asp +
Tyr169Thr + Ser207AspAsn 67Ser + Gly134Pro + Asn194Asp + Gly214Asp +
Arg218Asp + Ser221GluAsp 65Glu + Tyr137Asn + Thr211Pro + Arg218Asp +
Ile220Val + Ser221AspAsp 65Glu + Gly100Ser + Tyr104Ala + Ser207Glu +
Thr217Asp + Gly222AspAsp 98Glu + Asn 99Gln + Gln103Glu + Gly136Ser +
Leu209Asp + Arg218GluAsp 65Glu + Val 95Pro + Ser101Asp + Gln103Glu +
Asn168Ser + Ser219GluTyr104Val + Gly134Pro + Tyr137Asp + Ala164Glu +
Thr211Gly + Gly215SerSer101Asp + Gly135Pro + Ser190Asp + Ser191Asp +
Ser216Asp + Gly222GlnAsn 99Glu + Ser101Glu + Gln103Ser + Gly160Pro +
Ile213Asp + Gly222GluGly 68Ser + Ser138Glu + Ser139Glu + Gly160Glu +
Asn163Ser + Ser219GluArg 64Glu + Ser101Glu + Ser138Glu + Ser139Glu +
Asn161Ser + Thr206AsnGly 66Asn + Gly 68Asp + Tyr104Leu + Ser138Asp +
Ser139Asp + Ser207AspAsp 65Glu + Gln103Glu + Tyr137Cys + Thr206Asn +
Ser219Glu + Ile220GluLeu 96Asp+ Tyr137Asp + Leu209Thr + Ile213Gly +
Ser219Asp + Ile220GluLeu 96Asp + Asp 97Glu + Ser140Glu + Phe192Ala +
Thr217Asp + Gly222ProSer101Glu + Leu133Cys + Arg167Asp + Asn168Glu +
Leu209Ala + Ser216GluVal 95Gly + Thr106Glu + Arg167Asp + Asn168Glu +
Ser191Asp + Ile220ValGly 66Ser + Asp 97Glu + Tyr169Thr + Ser170Asp +
Thr217Glu + Arg218AspArg 64Asp + Ile107His + Gly135Asp + Gly136Asp +
Ile213Thr + Ser219AspAsp 97Glu + Leu133Thr + Gly134Glu + Arg218Glu +
Ser219Asp + Thr223SerAsp 98Glu + G1y102Ser + Leu133Met + Ser139Asp +
Ser140Asp + Ser216GluTyr137Ile + Ser139Glu + Ser140Glu + Gly160Gln +
Leu209Asp + Gly214AspLeu 96Ala + Asp 98Glu + Asn 99Glu + Asp165Glu +
Leu209Thr + Ser216GluAsn 99Gln + Ser138Asp + Asn161Glu + Gly215Glu +
Ser216Asp + Ile220GlySer105Asp + Thr106Pro + Gly136Pro + Gly160Glu +
Leu209Asp + Ser219AspGln103Glu + Ala166Thr + Asn194Glu + Leu209Asp +
Ser219Glu + Ile220CysTyr104Met + Thr106Ser + Ser139Asp + Thr206Pro +
Ser219Asp + Gly222GluIle107Gln + Asp165Glu + Asn168Glu + Ser191Asp +
Leu209Met + Ser219GluGly 68Ser + Thr106Glu + Ile107Gly + Asn162Glu +
Ser207Asp + Ile220AspArg 64Asp + Gly160Asp + Ser207Glu + Ile208Gly +
Ile213Leu + Ile220AspTyr137Asp + Ser170Asp + Phe192Ser + Ser207Glu +
Leu209Pro + Gly214GluTyr104Cys + Gly135Asp + Ser138Asp + Thr206Asp +
Gly214Gln + Arg218GluArg 64Asp + Asp 97Glu + Ser139Asp + Asn168Gln +
Thr206Asn + Gly214AspAsp 65Glu + Leu133Asn + Ser170Glu + Ile208Thr +
Ser216Glu + Arg218AspSer101Asp + Ser138Glu + Thr206Pro + Ser207Glu +
Ser219Glu + Ile220CysGly134Glu + Ser207Glu + Leu209Ser + Gly214Glu +
Ser219Glu + Ile220AlaArg 64Asp + Gly 68Asn + Gly135Asp + Gly136Gln +
Ser207Asp + Ser219AspSer101Asp + Ser138Glu + Ser207Asp + Leu209Ile +
Ser219Glu + Gly222GlnGly 66Asp + Ser105Asp + Ala164Gln + Phe192Ala +
Ser207Glu + Ser219AspArg 64Glu + Gly 66Asn + Asn 99Ser + Ser138Glu +
Ser207Asp + Ser219GluArg 64Asp + Ile107Thr + Ile213Gly + Ser216Asp +
Arg218Asp + Gly222GluGln103Glu + Ile107His + Gly135Pro + Gly136Asp +
Arg167Asp + Ser219GluGly 68Glu + Asp 97Glu + Thr106Glu + Phe192Met +
Ser207Glu + Ile213AlaAla164Glu + Asn168Ser + Ser191Glu + Ser216Asp +
Ser219Glu + Ile220ThrGly 68Gln + Asn162Glu + Ser190Asp + Leu209Thr +
Ser219Asp + Gly222GlnVal 95Cys + Ile107Asp + Tyr137Ala + Asn194Glu +
Gly214Asp + Ser216GluAsp 65Glu + Gly 70Gln + Leu209Gly + Ser216Glu +
Ser221Asp + Thr223GluSer101Glu + Gly205Gln + Leu209Glu + Ser216Asp +
Thr217Ser + Ile220ThrTyr137Thr + Ser139Glu + Leu209Asp + Gly215Pro +
Ser216Glu + Thr217GlnAsn 99Glu + Gly136Pro + Ser140Glu + Ser170Glu +
Asn194Glu + Ile220MetGly 66Gln + Asp 98Glu + Gly100Glu + Ser105Glu +
Ile220Asp + Thr223AsnAsp 98Glu + Gly100Glu + Ser105Asp + Gly136Ser +
Asn168Glu + Gly214ProVal 95Asn + Asp 97Glu + Ser101Asp + Asp165Glu +
Phe192His + Ile220GluGly135Glu + Asn163Asp + Phe192Tyr + Asn194Gln +
Ser219Glu + Ser221GluAsn 99Glu + Ser101Asp + Ser140Asp + Asn161Gln +
Ser190Asp + Gly215AsnGly 68Gln + Gly100Asp + Gln103Asp + Tyr137Gly +
Gly160Asp + Leu209CysGly100Asp + Gln103Asp + Gly160Gln + Leu209Asn +
Trp212Ser + Ser221GluGly135Asp + Ser138Asp + Asn162Gln + Leu209Val +
Ser216Glu + Thr223AspArg 64Asp + Gly 66Glu + Leu 96Thr + Thr106Ser +
Asn161Glu + Arg218GluTyr137Ser + Asn161Asp + Ser170Asp + Ser190Glu +
Leu209Asp + Gly215GlnLeu133Ala + Ser139Asp + Thr206Gln + Leu209Ala +
Ser216Asp + Ser219GluAsn 67Ser + Val 95Thr + Ser140Glu + Thr206Pro +
Ser216Glu + Ser219AspVal 95Asn + Ser138Glu + Ile208Gln + Leu209Val +
Ser216Glu + Ser219AspGly 70Asp + Asn 99Gln + Ser101Glu + Leu133Asn +
Leu209Gln + Ser216AspTyr104Gln + Ile107His + Ser138Asp + Ser191Asp +
Thr206Glu + Arg218AspThr106Glu + Ser138Glu + Thr206Gly + Ser219Glu +
Ile220Thr + Ser221GluTyr137Glu + Asn161Glu + Leu209Thr + Ile213Asp +
Ser216Asp + Thr217AsnArg 64Glu + Asn 67Glu + Ser170Asp + Trp212His +
Ile213Ala + Arg218GluVal 95Asp + Ile107Gly + Gly134Asp + Ser138Asp +
Asn161Gln + Ser190GluAsp 65Glu + Asp 98Glu + Gly160Ser + Asp165Glu +
Phe192Gly + Ser219GluAsp 97Glu + Leu133His + Gly136Glu + Ser138Asp +
Leu209Gly + Ser216AspAsp 65Glu + Ser138Glu + Tyr169Met + Ser207Asp +
Leu209Glu + Gly215ProLeu 96Gln + Asp 97Glu + Ser138Glu + Thr206Pro +
Ser207Glu + Leu209AspGly 70Gln + Asp 97Glu + Ser190Asp + Thr206Glu +
Ser216Asp + Ile220AsnArg 64Glu + Gly 66Pro + Gly 70Ser + Asp165Glu +
Tyr169Glu + Ser191AspGly136Asp + Asn163Asp + Phe192Pro + Ser216Glu +
Arg218Asp + Ile220ValGly 66Asp + Tyr137Gly + Asn162Ser + Ala164Glu +
Arg167Asp + Ser216GluGly100Asn + Gly102Asp + Tyr104His + Asp165Glu +
Ser216Asp + Arg218AspGly102Glu + Leu133Val + Ser140Glu + Thr206Glu +
Ile220Glu + Thr223ProSer105Glu + Leu133Asn + Ala164Asn + Asp165Glu +
Thr217Glu + Ser219AspGly 70Asp + Val 95Thr + Gly134Asp + Ser139Glu +
Asn161Glu + Asn194SerGly100Ser + Gly135Glu + Ser140Glu + Asn161Asp +
Ile213Cys + Arg218GluGly136Asp + Tyr137Gly + Ser140Asp + Asn162Glu +
Thr211Gly + Gly214AspVal 95Gly + Tyr104Ala + Ser138Glu + Asn163Asp +
Arg167Asp + Ser219AspAsp 65Glu + Gly 70Asn + Val 95Ser + Ser191Asp +
Thr206Glu + Ser216AspTyr104Leu + Leu133Cys + Gly134Asp + Asn162Asp +
Tyr169Pro + Ser216AspVal 95Glu + Asn 99Asp + Tyr104Ala + Tyr137Ala +
Leu209Asp + Ile220CysGly 66Glu + Thr106Ser + Leu209Gly + Gly215Asn +
Ser216Asp + Ile220HisThr 71Pro + Gly102Glu + Gly134Asp + Ser139Asp +
Ile220Pro + Ser221AspLeu 96Ser + Asp 98Glu + Ser101Asp + Gln103Ser +
Gly160Asp + Ser216GluLeu 96Cys + Ala164Asn + Tyr169Pro + Thr217Asn +
Arg218Asp + Gly222AspTyr104Asn + Gly136Glu + Tyr137His + Ser140Glu +
Ser216Glu + Ile220CysAsp 98Glu + Gly135Asp + Ser138Glu + Asn163Glu +
Phe192Cys + Ile220SerGly 66Glu + Asn 99Glu + Gly102Pro + Thr106Asn +
Ser139Glu + Ser221AspGly 68Glu + Tyr104Ala + Ala164Gln + Asn168Asp +
Ser219Glu + Thr223AsnAsh 99Glu + Gly102pro + Ser140Asp + Ser207Glu +
Leu209Asn + Gly222AspSer105Asp + Thr106Gln + Gly160Asp + Ser191Asp +
Phe192His + Gly203GlnAsp 98Glu + Tyr104Thr + Asn162Gln + Arg167Asp +
Ser170Glu + Ser216GluAsn 99Asp + Gly102Ser + Tyr137Asp + Asp165Glu +
Ile220Ala + Thr223GlyAsp 98Glu + Thr106Pro + Gly135Asp + Ser138Glu +
Phe192Asp + Ile220GlnArg 64Glu + Val 95Ala + Leu 96Asn + Ala164Glu +
Leu209Ala + Gly214GluArg 64Glu + Gly136Asn + Ala166Ser + Leu209His +
Gly214Asp + Ser221GluAsn 67Asp + Gly102Asn + Ala164Glu + Asn168Glu +
Ser207Asp + Ile220LeuGly135Glu + Gly136Gln + Ser139Asp + Gly160Asn +
Ala164Ser + Thr206GluSer105Asp + Asn163Gln + Thr206Glu + Gly215Glu +
Arg218Glu + Ile220CysGly 68Gln + Ser101Asp + Ser139Glu + Asn162Asp +
Asp165Glu + Leu209GlnGly100Ser + Ser140Asp + Asn162Glu + Asp165Glu +
Pro204Gln + Ser207GluThr106Glu + Ile107Ala + Gly136Glu + Ser140Asp +
Asn168Gln + Ser219AspArg 64Glu + Val 95Thr + Asp 98Glu + Tyr104Thr +
Ser140Glu + Ser191AspII.洗涤组合物
在本发明的另一个实施方案中，在用于清洁需要除去含蛋白污渍的多种表面的组合物中含有有效量的一种或多种酶变异体。这些洗涤组合物包括用于清洁硬表面的洗涤剂组合物，形式不限(如液体和粒状)；用于清洁织物的洗涤剂组合物，形式不限(如粒状、液体和棒状)；洗餐具用组合物(形式不限)；口腔清洁组合物，形式不限(如牙粉，牙膏和漱口药制品)；假牙清洁组合物，形式不限(如液体，片状)；和隐形眼镜清洁组合物，形式不限(如液体，片状)。
洗涤组合物中除了含有上文所述的蛋白酶K变异体外，还含有与这些蛋白酶相容的一种或多种洗涤组合物成份。这里用到的“洗涤组合物成份”指那些为所希望的清洗组合物的特定类型和产品形式(如液体、粒状、棒状、喷雾型、杆状、糊状、胶状)而选择的任何液体、固体或气体物质，这些物质也与组合物中所用到的蛋白酶K相容。洗涤组合物成份的具体选择考虑到使用时(如通过洗涤剂使用)被清洁的表面材料和适合清洁条件的组合物形式而容易被作到。这里用到的“相容”指洗涤组合物成份不会将蛋白酶K变异体的蛋白水解活性降低到在正常使用情况下蛋白酶失去所需效果的程度。一些具体的洗涤组合物成份在后面详细地举例说明。
这里用到的“酶变异体的有效量”指酶变异体在具体的洗涤组合物中达到需要的酶活性所需的量。这种有效量很容易为本领域的技术人员确定，其基于多种因素，如所用的具体的酶变异体、洗涤作用、洗涤组合物中特殊组份、是否需要湿或干(如粒状、棒状)的组合物等等。优选的洗涤组合物包含约0.0001％-10％的本发明中的一种或多种酶变异体，更优选约0.001％-1％，最优选约0.01％-0.1％。其中加入了酶变异体的各种洗涤组合物的几个实例将在后面更详细地讨论。这里所用到的所有份数、百分比和比率除非另外说明，均按重量计。
这里用到的“非织物洗涤组合物”包括硬表面洗涤组合物、洗餐具用组合物、口腔清洁组合物、假牙清洁组合物和隐形眼镜清洁组合物。A.用于硬表面、餐具和织物的洗涤组合物
本发明中的酶变异体可用于多种洗涤组合物中，这些洗涤组合物具有丰富的泡沫和优良的不溶底物去除能力。因此这些酶变异体可与各种常规组分合用得到全配制的硬表面清洁剂、餐具清洗组合物、洗涤织物用组合物等等。这些组合物可为液体、粒状、棒状等多种形式。这些组合物可配制为现代的“浓缩”洗涤剂形式，其中包含了高达30％-60％(重量)的表面活性剂。
这些洗涤组合物可任选地和优选地包含各种阴离子型的、非离子型的、两性离子型的等表面活性剂。这些表面活性剂在组合物中的典型含量约为5％-35％。
这里有用的表面活性剂的非限制性实例包括常规的C11-C18烷基苯磺酰盐和伯和无规型烷基硫酸盐，具有式：CH3(CH2)x(CHOSO3)-M+)CH3和CH3(CH2)y(CHOSO3 -M+)CH2CH3的C10-C18仲(2，3)烷基硫酸盐，其中x和y+1为至少约7的整数，优选至少约9的整数，M为水溶性的阳离子，尤其是钠离子，C10-C18烷基烷氧基硫酸盐(特别是EO1-5乙氧基硫酸盐)，C10-C18烷基烷氧基羧酸盐(特别是EO1-5乙氧基羧酸盐)，C10- C18烷基多苷及其相应的硫酸化多苷，C12-C18α-磺化的脂肪酸酯，C12-C18烷基和烷基酚烷氧基化物(特别是乙氧基化物和混合的乙氧基/丙氧基化物)，C12-C18甜菜碱和磺化甜菜碱(“Sultaines”)，C10-C18胺氧化物等等。在此烷基烷氧基硫酸盐(AES)和烷基烷氧基羧酸盐(AEC)是优选的。(根据配制者的需要，这些表面活性剂与上面的胺氧化物和/或甜菜碱或磺化甜菜碱表面活性剂联合使用也是优选的)。其它常规的有用的表面活性剂列于标准教科书中。特别有用的表面活性剂包括C10-C18N-甲基葡萄糖酰胺，其公开于Connor等人的美国专利5,194,639，1993年3月16日授权，在此引入作为参考。
洗涤组合物中的多种其它有用组分也能包括在这里的组合物中，包括其它活性成份、载体、水溶助长剂、加工助剂、染料或色素、用于液体配方中的溶剂等。如果需要另外增加泡沫，增泡剂如C10-C16烷醇酰胺可加到组合物中，一般为约1％-10％的量。 C10-C14单乙醇胺和二乙醇酰胺为典型的这类增泡剂。这些增泡剂与多泡的附加表面活性剂如上面提到的胺氧化物、甜菜碱和磺化甜菜碱合用也有很好的效果。如果需要，可加入可溶的镁盐如MgCl2、MgSO4等，以提供更多的泡沫，加入的量一般为约0.1％-2％。
这里的液体洗涤组合物可以含水和其它溶剂作为载体。低分子量的伯或仲醇，如甲醇、乙醇、丙醇和异丙醇都是合适的。对可溶性表面活性剂，一元醇是优选的，但多元醇如那些含大约2到6个碳原子并含大约2到6个羟基的多元醇(如1，3-丙二醇、乙二醇、甘油和1，2-丙二醇)也能使用。组合物中可含有约5％-90％的这些载体，一般约含10％-50％的载体。
这里的洗涤组合物优选被配制成在水洗操作时，洗涤水的pH控制在约6.8-11.0之间。因而一般将最终产品配制在这个范围内。将pH控制在建议使用值的技术包括使用缓冲剂、碱、酸等，对本领域的技术人员来说它们是熟知的。
当配制本发明中的硬表面洗涤组合物和织物洗涤组合物时，配制者也许希望加入约5％-50％(重量)的各种助洗剂。一般的助洗剂包括1-10微米的沸石、多羧酸盐如柠檬酸盐和氧联二琥珀酸盐、层状硅酸盐、磷酸盐等。其它常规助洗剂列于标准的配方书中。
另外，配制者可能也希望在组合物中使用各种附加酶，如纤维素酶、脂肪酶、淀粉酶和蛋白酶，常用量为约0.001％-1％(重量)。在洗衣洗涤剂领域，许多有清洁能力又能护理织物的酶已广为人知。
许多漂白化合物，如过碳酸盐和过硼酸盐等也用于这些组合物中，一般含量为约1％-15％(重量)。如果需要，这些组合物中也可包含漂白活化剂，如四乙酰乙二胺、壬酰氧基苯磺酸盐等，它们已为本领域所熟知。常用量为约1％-10％(重量)。
各种污垢解脱剂如阴离子型低聚酯、各种螯合剂，尤其是氨基膦酸盐和乙二胺二琥珀酸盐、各种粘土类去污剂，尤其是乙氧基化四亚乙基戊胺、各种分散剂尤其是聚丙烯酸酯和聚天冬氨酸、各种增白剂尤其是阴离子型增白剂、各种抑泡剂尤其是聚硅氧烷和仲醇、各种织物柔软剂尤其是绿土等都能用于这些组合物，其含量为约1％-35％(重量)。标准的配方书和已公开的专利中均有这些常规物质的全面详细的描述。
酶稳定剂也被用于这些洗涤组合物中。这些酶稳定剂包括丙二醇(优选约1％-10％)，甲酸钠(优选约0.1％-1％)和甲酸钙(优选约0.1％- 1％)。1.硬表面洗涤组合物
这里所说的“硬表面洗涤组合物”指用于清洁硬表面如地板、墙、浴室贴面等的液体和粒状的洗涤组合物。本发明中的硬表面洗涤组合物包含有效量的一种或多种本发明的酶变异体，组合物中所含活性酶的重量优选约0.001-10％，更优选约0.01％-5％，最优选约0.05％-1％。除含一种或多种酶变异体外，这些硬表面洗涤组合物一般还包括表面活性剂和水溶性多价螯合助洗剂。在某些特殊的产品如窗户清洁喷雾剂中，有时不用表面活性剂，因为它们可能在玻璃表面上产生薄膜状或条纹状的残余物。
表面活性剂组分，如果有的话，只占本发明的组合物的0.1％，而通常的组合物将含约0.25％-10％，优选约1％-5％。
通常组合物含有约0.5％-50％的洗涤助洗剂，优选约1％-10％。
优选的pH值应在约8-12范围内，如果需要调整，可以使用常规的pH调整试剂如氢氧化钠、碳酸钠或盐酸。
组合物中可以包含溶剂。常用的溶剂包括，但不仅限于乙二醇醚，如二乙二醇单己醚、二乙二醇单丁醚、乙二醇单丁醚、乙二醇单己醚、丙二醇单丁醚、二丙二醇单丁醚，和二元醇，如2，2，4-三甲基-1，3-戊二醇和2-乙基-1，3-己二醇。当使用这些溶剂时，它们的含量约为0.5％-15％，优选约3％-11％。
另外，高挥发性溶剂如异丙醇或乙醇也用于本发明的组合物中，当组合物已完全应用于表面而没有冲掉时，它们可使之快速蒸发掉。当使用时，挥发性溶剂常常在组合物中占约2％-12％。
本发明中硬表面洗涤组合物的实施方案在下面的实施例中列表说明。
实施例7-12
液体硬表面洗涤组合物
实施例序号组份 7 8 9 10 11 12Asn67Ser 0.05 0.50 0.02 0.03 0.10 0.03Val95His - - - - 0.20 0.02Na2DIDA*EDTA** - - 2.90 2.90 - -柠檬酸钠 - - - - 2.90 2.90C12烷基苯磺酸钠 1.95 - 1.95 - 1.95 -C12烷基硫酸钠 - 2.20 - 2.20 - 2.20C12(乙氧基)***硫酸钠 - 2.20 - 2.20 - 2.20C12二甲胺氧化物 - 0.50 - 0.50 - 0.50枯烯磺酸钠 1.30 - 1.30 - 1.30 -己基卡必醇*** 6.30 6.30 6.30 6.30 6.30 6.30水**** 平衡到100％*N-二乙二醇 N，N-亚胺基二乙酸二钠**乙二胺二乙酸四钠***二乙二醇单己醚****所有配方调至pH7
在实施例7-10中，用表2-36中列出的其它蛋白酶K变异体替换Asn 67 Ser具有大致相同的结果。
在实施例11-12中，表2-36中列出的蛋白酶K变异体的其它任何组合代替Asn67Ser和Val95His，具有大致相同的结果。
实施例13-18
清洁硬表面和除去室内霉菌的喷雾型组合物
实施例序号组份 13 14 15 16 17 18Gly134Asn+Ser140Asp 0.50 0.05 0.60 0.30 0.20 0.30Leu96Gly+Leu209Pro - - - - 0.30 0.10辛基硫酸钠 2.00 2.00 2.00 2.00 2.00 2.00十二烷基硫酸钠 4.00 4.00 4.00 4.00 4.00 4.00氢氧化钠 0.80 0.80 0.80 0.80 0.80 0.80硅酸钠 0.04 0.04 0.04 0.04 0.04 0.04香料 0.35 0.35 0.35 0.35 0.35 0.35水 平衡至100％产品pH约为7。
在实施例13-16中，用表2-36中列出的其它蛋白酶K变异体代替Gly134Asn+Ser140Asp，具有大致相同的结果。
在实施例17-18中，用表2-36中列出的其它蛋白酶K变异体的任何组合代替Gly134Asn+Ser140Asp和Leu96Gly+Leu209Pro，具有大致相同的结果。2.洗餐具用洗涤组合物
在本发明的另一个实施方案中，洗餐具用洗涤组合物包含一种或多种本发明的酶变异体。这里用到的“洗餐具用组合物”指所有形式的用于洗涤餐具的组合物，包括但不限于粒状和液体形式的洗涤剂。本发明中洗餐具用洗涤组合物的实施方案在下面的实施例中列表说明。
实施例19-24
洗餐具用洗涤组合物
实施例序号
19 20 21 22 23 24Ala164Asp 0.05 0.50 0.02 0.40 0.10 0.03Ser191Asp+Phe192Met
+Asn194Gln - - - - 0.40 0.02C12-C14 N-甲基-葡糖酰胺 0.90 0.90 0.90 0.90 0.90 0.90C12乙氧基(1)硫酸盐 12.00 12.00 12.00 12.00 12.00 12.002-甲基十一烷酸 4.50 4.50 4.50 4.50 4.50 4.50C12乙氧基(2)羧酸盐 4.50 4.50 4.50 4.50 4.50 4.50C12醇乙氧基化物(4) 3.00 3.00 3.00 3.00 3.00 3.00C12胺氧化物 3.00 3.00 3.00 3.00 3.00 3.00枯烯磺酰钠 2.00 2.00 2.00 2.00 2.00 2.00乙醇 4.00 4.00 4.00 4.00 4.00 4.00Mg++(如MgCl2) 0.20 0.20 0.20 0.20 0.20 0.20Ca++(如CaCl2) 0.40 0.40 0.40 0.40 0.40 0.40水 平衡至100％产品pH调至7。
在实施例19-22中，用表2-36列出的其它蛋白酶K变异体代替Ala164Asp，具有大致相同的结果。
在实施例23-24中，用表2-36列出的其它蛋白酶K变异体的任何组合代替A1al64Asp和Ser191Asp+Phe192Met+Asn194Gln，具有大致相同的结果。3.织物洗涤组合物
本发明的另一个实施方案中，织物洗涤组合物包含本发明的一种或多种酶变异体。这里用到的“织物洗涤组合物”指所有形式的用于洗涤织物的洗涤组合物，包括但不限于粒状、液体形式的和棒状的洗涤剂。优选的织物洗涤组合物是液体形式的。a.粒状织物洗涤组合物
本发明中的粒状织物洗涤组合物含有有效量的本发明中的一种或多种酶变异体，优选活性酶占组合物重量的大约0.001％-10％，更优选占大约0.005％-5％，最优选占大约0.01％-1％。除了一种或多种酶变异体外，粒状织物洗涤组合物还常常含有至少一种表面活性剂，一种或多种助洗剂，在某些情况下包括漂白剂。
本发明中粒状织物洗涤组合物的实施方案在下面的实施例中详细说明。
实施例25-28
粒状织物洗涤组合物
实施例序号组份 25 26 27 28Ile208Ala 0.10 0.20 0.03 0.05Trp212Phe - - 0.02 0.05C13直链烷基苯磺酸盐 22.00 22.00 22.00 22.00磷酸盐(如三聚磷酸钠) 23.00 23.00 23.00 23.00碳酸钠 23.00 23.00 23.00 23.00硅酸钠 14.00 14.00 14.00 14.00沸 8.20 8.20 8.20 8.20螯合剂(二亚乙基三胺五乙酸) 0.40 0.40 0.40 0.40硫酸钠 5.50 5.50 5.50 5.50水 平衡到100％
在实施例25-26中，用表2-36列出的其它蛋白酶K变异体代替Ile208Ala，具有大致相同的结果。
在实施例27-28中，用表2-36列出的其它蛋白酶K变异体的任何组合代替Ile208Ala和Trp212Phe，具有大致相同的结果。
实施例29-32
粒状织物洗涤组合物
实施例序号组份 29 30 31 32Asnl94Glu+Leu209Gly 0.10 0.20 0.03 0.05Gly100Asn+Gln103Ser+Ser207Asp - - 0.02 0.05
+Leu209ValC12烷基苯磺酸盐 12.00 12.00 12.00 12.00沸石A(1-10微米) 26.00 26.00 26.00 26.002-丁基辛酸 4.00 4.00 4.00 4.00C12-C14仲(2，3)烷基硫酸钠 5.00 5.00 5.00 5.00荧光增白剂 0.10 0.10 0.10 0.10柠檬酸钠 5.00 5.00 5.00 5.00硫酸钠 17.00 17.00 17.00 17.00水和次要成份 平衡到100％
在实施例29-30中，用表2-36列出的其它蛋白酶K变异体代替Asn194Glu+Leu209Gly，具有大致相同的结果。
在实施例31-32中，用表2-36列出的其它蛋白酶K变异体的任何组合代替Asn194Glu+Leu209Gly和Gly100Asn+Gln103Ser+Ser207Asp+Leu209Valo，具有大致相同的结果。
实施例33-36
粒状织物洗涤组合物
实施例序号成份 33 34 35 36Arg64Asp+Gly70Gln+Thr71Gly 0.10 0.20 0.03 0.05Phe192Asp+Asn194Ser - - 0.02 0.05C13直链烷基苯磺酸盐 22.00 22.00 22.00 22.00磷酸盐(如三聚磷酸钠) 23.00 23.00 23.00 23.00碳酸钠 23.00 23.00 23.00 23.00硅酸钠 14.00 14.00 14.00 14.00沸石 8.20 8.20 8.20 8.20螯合剂(二亚乙基三胺五乙酸) 0.40 0.40 0.40 0.40硫酸钠 5.50 5.50 5.50 5.50水 平衡至100％
在实施例33-34中，用表2-36列出的其它蛋白酶K变异体代替Arg64Asp+Gly70Gln+Thr71Gly，具有大致相同的结果。
在实施例35-36中，用表2-36列出的其它蛋白酶K变异体的任何组合代替Arg64Asp+Gly70Gln+Thr71Gly和Phe192Asp+Asn194Ser，具有大致相同的结果。
实施例37-40
粒状织物洗涤组合物
实施例序号组份 37 38 39 40Ala164His+Ala166Gly 0.10 0.20 0.03 0.05Ile213Pro+Ser216Glu+Ile220Glu - - 0.02 0.05C12烷基苯磺酸盐 12.00 12.00 12.00 12.00沸石A(1-10微米) 26.00 26.00 26.00 26.002-丁基辛酸 4.00 4.00 4.00 4.00C12-C14仲(2，3)烷基硫酸，钠盐5.00 5.00 5.00 5.00柠檬酸钠 5.00 5.00 5.00 5.00荧光增白剂 0.10 0.10 0.10 0.10硫酸钠 17.00 17.00 17.00 17.00水及次要成份 平衡至100％
在实施例37-38中，用表2-36列出的其它蛋白酶K变异体代替Ala164His+Ala166Gly，具有大致相同的结果。
在实施例39-40中，用表2-36列出的其它蛋白酶K变异体的任何组合代替Ala164His+Ala166Gly和Ile213Pro+Ser216Glu+Ile220Glu，具有大致相同的结果。
实施例41-42
粒状织物洗涤组合物
实施例序号组份 41 42直链烷基苯磺酸盐 11.4 10.70牛油烷基硫酸盐 1.80 2.40C14-C15烷基硫酸盐 3.00 3.10C14-C15醇七倍乙氧化物 4.00 4.00牛油醇11倍乙氧化物 1.80 1.80分散剂 0.07 0.1聚硅氧烷流体 0.80 0.80柠檬酸三钠 14.00 15.00柠檬酸 3.00 2.50沸石 32.50 32.10马来酸丙烯酸共聚物 5.00 5.00二亚乙基三胺五亚甲基膦酸 1.00 0.20Gly203Gln+Thr211Gly+Ile213Leu+Gly214Asn 0.30 0.30脂肪酶 0.36 0.40淀粉酶 0.30 0.30硅酸钠 2.00 2.50硫酸钠 3.50 5.20聚乙烯吡咯烷酮 0.30 0.50过硼酸盐 0.5 1苯酚磺酸盐 0.1 0.2过氧化物酶 0.1 0.1次要成份 加至100 加至100
实施例43-44
粒状织物洗涤组合物
实施例序号组份 43 44直链C12烷基苯磺酸钠 6.5 8.0硫酸钠 15.0 18.0沸石A 26.0 22.0次氮基三乙酸钠 5.0 5.0聚乙烯吡咯烷酮 0.5 0.7四乙酰乙二胺 3.0 3.0硼酸 4.0 -过硼酸盐 0.5 1苯酚磺酸盐 0.1 0.2Asp98Glu+Tyr104Leu 0.4 0.4填料(如硅酸盐；碳酸盐；香料；水) 加至100 加至100
实施例45
致密粒状织物洗涤组合物组份 重量％烷基硫酸盐 8.0烷基乙氧基硫酸盐 2.0C25和C45醇3和7倍乙氧化物混合物 6.0多羟基脂肪酸酰胺 2.5沸石 17.0层状硅酸盐/柠檬酸盐 16.0碳酸盐 7.0马来酸丙烯酸共聚物 5.0污垢解脱聚合物 0.4羧甲基纤维素 0.4聚(4-乙烯基吡啶)-N-氧化物 0.1乙烯基咪唑和乙烯基吡咯烷酮的共聚体 0.1PEG 2000 0.2Gly136Ser+Tyr137Met+Ser138Glu 0.5脂肪酶 0.2纤维素酶 0.2四乙酰乙二胺 6.0过碳酸盐 22.0亚乙基二胺二琥珀酸 0.3抑泡剂 3.54，4′-双(2-吗啡代-4-苯胺基-S-三吖嗪-6-基 0.25氨基)芪-2，2′-二磺酸二钠4，4′-双(2-硫代styril)联苯二钠 0.05水，香料及次要组份 加至100
实施例46
粒状织物洗涤组合物组份 重量％直链烷基苯磺酸盐 7.6C16-C18烷基硫酸盐 1.3C14-C15醇7倍乙氧化物 4.0椰子烷基二甲基羟乙基铵氯化物 1.4分散剂 0.07聚硅氧烷流体 0.8柠檬酸三钠 5.0沸4A 15.0马来酸丙烯酸共聚体 4.0二亚乙基三胺五亚甲基膦酸 0.4过硼酸盐 15.0四乙酰乙二胺 5.0绿土 10.0聚(氧乙烯)(MW 300,000) 0.3Ser219Asp 0.4脂肪酶 0.2淀粉酶 0.3纤维素酶 0.2硅酸钠 3.0碳酸钠 10.0羧甲基纤维素 0.2增白剂 0.2水、香料和次要组份 加至100
实施例47
粒状织物洗涤组合物组份 重量％直链烷基苯磺酸盐 6.92牛油烷基硫酸盐 2.05C14-C15醇7倍乙氧化物 4.4C12-C15烷基乙氧基硫酸盐-3倍乙氧化物 0.16沸石 20.2柠檬酸盐 5.5碳酸盐 15.4硅酸盐 3.0马来酸丙烯酸共聚物 4.0羧甲基纤维素酶 0.31污垢解脱聚合物 0.30Ser190Glu+Ser191Glu+Phe192Leu+Asn194Ser 0.2脂肪酶 0.36纤维素酶 0.13过硼酸盐四水合物 11.64过硼酸盐一水合物 8.7四乙酰乙二胺 5.0二亚乙基三胺五亚甲基膦酸 0.38硫酸镁 0.40增白剂 0.19香料、聚硅氧烷、抑泡剂 0.85次要组份 加至100b.液体织物洗涤组合物
本发明的液体织物洗涤组合物含有有效量的本发明中的一种或多种酶变异体，活性酶的含量优选约占组合物重量的0.005％-5％，更优选约占0.01％-1％。这些液体织物洗涤组合物一般还包含阴离子型表面活性剂、脂肪酸、水溶性洗涤助洗剂及水。
本发明中液体织物洗涤组合物的实施方案在下面的实施例中详细说明。
实施例48-52
液体织物洗涤组合物
实施例序号组份 48 49 50 51 52Ile220Asp 0.05 0.03 0.30 0.03 0.10Thr206Asp+Ile220Met - - - 0.01 0.20C12-C14烷基硫酸钠 20.00 20.00 20.00 20.00 20.002-丁基辛酸 5.00 5.00 5.00 5.00 5.00柠檬酸钠 1.00 1.00 1.00 1.00 1.00C10醇乙氧基化物(3) 13.00 13.00 13.00 13.00 13.00单乙醇胺 2.50 2.50 2.50 2.50 2.50水/丙二醇/乙醇(100∶1∶1) 平衡到100％
在实施例48-50中，用表2-36列出的其它蛋白酶K变异体代替Ile220Asp，具有大致相同的结果。
在实施例51-52中，用表2-36列出的其它蛋白酶K变异体的任何组合代替Ile220Asp和Thr206Asp+Ile220Met，具有大致相同的结果。
实施例53-57
液体织物洗涤组合物
实施例序号组份 53 54 55 56 57Gly136Ser 0.05 0.03 0.30 0.03 0.10Gly215Pro - - - 0 01 0.20C12-C14烷基硫酸钠 20.00 20.00 20.00 20.00 20.002-丁基辛酸 5.00 5.00 5.00 5.00 5.00柠檬酸钠 1.00 1.00 1.00 1.00 1.00C10醇乙氧基化物(3) 13.00 13.00 13.00 13.00 13.00单乙醇胺 2.50 2.50 2.50 2.50 2.50水/丙二醇/乙醇(100∶1∶1) 平衡至100％
在实施例53-55中，用表2-36列出的其它蛋白酶K变异体代替Gly136Ser，具有大致相同的结果。
在实施例56-57中，用表2-36列出的其它蛋白酶K变异体的任何组合代替Gly136Ser和Gly215Pro，具有大致相同的结果。
实施例58-59
液体织物洗涤组合物
实施例序号组份 58 59C12-C14链烯基琥珀酸 3.0 8.0柠檬酸一水合物 10.0 15.0C12-C15烷基硫酸钠 8.0 8.0C12-C15醇2倍乙氧化物硫酸钠 - 3.0C12-C15醇7倍乙氧化物 - 8.0C12-C15醇5倍乙氧化物 8.0 -二亚乙基三胺五(亚甲基膦酸) 0.2 -油酸 1.8 -乙醇 4.0 4.0丙二醇 2.0 2.0Pro204Asn+Thr211Pro+Gly214Gln 0.2 0.2
+Gly215Glu+Thr217Ser聚乙烯吡咯烷酮 1.0 2.0抑泡剂 0.15 0.15NaOH 调pH至7.5过硼酸盐 0.5 1苯酚磺酸盐 0.1 0.2过氧化物酶 0.4 0.1水及次要组份 加至100份
在实施例58和59中，用表2-36列出的其它蛋白酶K变异体代替Pro204Asn+Thr211Pro+Gly214Gln+Gly215Glu+Thr217Ser，均具有大致相同的结果。
实施例60-62
液体织物洗涤组合物
实施例序号组份 60 61 62柠檬酸 7.10 3.00 3.00脂肪酸 2.00 - 2.00乙醇 1.93 3.20 3.20硼酸 2.22 3.50 3.50单乙醇胺 0.71 1.09 1.091，2丙二醇 7.89 8.00 8.00枯烯磺酸钠 1.80 3.00 3.00甲酸钠 0.08 0.08 0.08NaOH 6.70 3.80 3.80硅抗泡剂 1.16 1.18 1.18Leu96Met+Asn99Ser+Ser105Asp 0.0145 - -Thr211Ser+Gly222Asp - 0.0145 -Tyr169Ser+Ser190Asp+Ile220Thr - - 0.0145脂肪酶 0.200 0.200 0.200纤维素酶 - 7.50 7.50污垢解脱聚合物 0.29 0.15 0.15抗泡剂 0.06 0.085 0.085增白剂36 0.095 - -增白剂3 - 0.05 0.05C12烷基苯磺酸 9.86 - -C12-C15烷基聚乙氧化物(2.5)硫酸盐 13.80 18.00 18.00C12葡萄糖酰胺 - 5.00 5.00C12-C13烷基聚乙氧化物(9) 2.00 2.00 2.00水、香料及次要组分 平衡至100％C.棒状织物洗涤组合物
本发明中适用于手洗脏的织物的棒状织物洗涤组合物含有有效量的本发明的一种或多种酶变异体，优选占组合物重量的大致0.001％-10％，更优选占大约0.01％-1％。
本发明中棒状织物洗涤组合物的实施方案在下面的实施例中详细说明。
实施例63-66
棒状织物洗涤组合物
实施例序号组份 63 64 65 66Leu96Ala+Asp97Glu+Ser101Asp 0.3 - 0.1 0.02
+Ile107AspSer193Asp - - 0.4 0.03C12-C16烷基硫酸钠 20.0 20.0 20.0 20.00C12-C14 N-甲基葡萄糖酰胺 5.0 5.0 5.0 5.00C11-C13烷基苯磺酰钠 10.0 10.0 10.0 10.00碳酸钠 25.0 25.0 25.0 25.00焦磷酸钠 7.0 7.0 7.0 7.00三聚磷酸钠 7.0 7.0 7.0 7.00沸石A(0.1-10μ) 5.0 5.0 5.0 5.00羧甲基纤维素 0.2 0.2 0.2 0.20聚丙烯酸酯(MW 1400) 0.2 0.2 0.2 0.20椰子单乙醇酰胺 5.0 5.0 5.0 5.00增白剂、香料 0.2 0.2 0.2 0.20CaSO4 1.0 1.0 1.0 1.00MgSO4 1.0 1.0 1.0 1.00水 4.0 4.0 4.0 4.00填料* 平衡到100％*可从常用的材料如CaCO3、滑石粉、粘土、硅酸盐等中选择。
在实施例63-64中，用表2-36列出的蛋白酶K变异体代替Leu96Ala+Asp97Glu+Ser101Asp+Ile107Asp，具有大致相同的结果。
在实施例65-66中，用表2-36列出的其它蛋白酶K变异体的任何组合代替Leu96Ala+Asp97Glu+Ser101Asp+Ile107Asp和Ser193Asp，具有大致相同的结果。
实施例67-70
棒状织物洗涤组合物
实施例序号组份 67 68 69 70Arg64Asp+Gly68Gln 0.3 - 0.1 0.02Tyr137Gln - 0.3 0.4 0.03C12-C16烷基硫酸钠 20.0 20.0 20.0 20.00C12-C14N-甲基葡萄糖酰胺 5.0 5.0 5.0 5.00C11-C13烷基苯磺酸钠 10.0 10.0 10.0 10.00碳酸钠 25.0 25.0 25.0 25.00焦磷酸钠 7.0 7.0 7.0 7.00三聚磷酸钠 7.0 7.0 7.0 7.00沸石A(0.1-10μ) 5.0 5.0 5.0 5.00羧甲基纤维素 0.2 0.2 0.2 0.20聚丙烯酸酯(MW 1400) 0.2 0.2 0.2 0.20椰子单乙醇酰胺 5.0 5.0 5.0 5.00增白剂、香料 0.2 0.2 0.2 0.20CaSO4 1.0 1.0 1.0 1.00MgSO4 1.0 1.0 1.0 1.00水 4.0 4.0 4.0 4.00填料* 平衡到100％*可从常规的材料如CaCO3、滑石粉、粘土、硅酸盐等中选择。
在实施例67中，用表2-36列出的其它蛋白酶K变异体代替Arg64Asp+Gly68Gln，具有大致相同的结果。
在实施例68中，用表2-36列出的其它蛋白酶K变异体代替Tyr137Gln，具有大致相同的结果。
在实施例69-70中，用表2-36列出的其它蛋白酶K变异体的任何组合代替Arg64Asp+Gly68Gln和Tyr137Gln，具有大致相同的结果。B.其它洗涤组合物
除了上述已讨论的硬表面，餐具和纤维洗涤组合物外，本发明中的一种或多种酶变异体也可掺入到需要水解不溶底物的多种其它清洗组合物中。这些其它的清洗组合物包括但不仅限于口腔清洁组合物、假牙清洗组合物和隐形眼镜清洗组合物。1.口腔清洁组合物
在本发明的另一个实施方案中，可药用量的本发明的一种或多种酶变异体加入到组合物中，用以去除牙或假牙中的蛋白质类污垢。这里用到的“口腔清洁组合物”指洗牙水、牙膏、牙胶、牙粉、漱口药、口腔喷雾剂、口胶、咀嚼胶、锭剂、香粉、药片、生物胶、预防膏、假牙处理液等。优选按重量计口腔清洁组合物中含约0.0001％-20％的本发明的一种或多种酶变异体，更优选含约0.001％-10％，最优选含约0.01％-5％，和一种可药用的载体。这里用到的“可药用”指那些用术语描述的药品、药物或惰性成份适合与人和较低等动物的组织直接接触使用而没有不当的毒性、不适应性、不稳定性、刺激性、过敏反应等，同时具有合理的益处/风险比。
通常，口腔清洁组合物中口腔清洁成份中的可药用的口腔清洁载体成份通常占组合物重量的大约50％-99.99％，优选占大约65％-99.99％，更优选占大约65％-99％。
本发明中的口腔清洁组合物中可能包含的可药用的载体和任选成份对本领域的技术人员来说是熟知的。这些口腔清洁组合物中用到的多种组合物类型、载体成份和任选成份，公开于以下专利文献中：美国专利5,096,700，Seibel，1992年3月17日授权；美国专利5,028,414，Sampathkumar，1991年7月2日授权；和美国专利5,028,415,Benedict，Bush和Sunberg，1991年7月2日授权；所有这些在此引入作为参考。
本发明中口腔清洁组合物的实施方案在下面的实施例中详细说明。
实施例71-74
洗牙水组合物
实施例序号组份 71 72 73 74Gly160Pro 2.000 3.500 1.500 2.000山梨醇(70％水溶液) 35.00 35.000 35.000 35.000PEG-6* 1.000 1.000 1.000 1.000二氧化硅牙磨粉** 20.000 20.000 20.000 20.000氟化钠 0.243 0.243 0.243 0.243二氧化钛 0.500 0.500 0.500 0.500糖精钠 0.286 0.286 0.286 0.286烷基硫酸钠(27.9％水溶液) 4.000 4.000 4.000 4.000香料 1.040 1.040 1.040 1.040羟乙烯基聚合物*** 0.300 0.300 0.300 0.300藻胶**** 0.800 0.800 0.800 0.800水 平衡至100％*PEG-6＝分子量为600的聚乙二醇**由J.M.Huber提供的确定为Zeodent119的沉淀二氧化硅。***Carbopol，由B.F.Goodrich化学公司提供。****Iota藻胶，由Hercules化学公司提供。
在实施例71-74中，用表2-36列出的其它蛋白酶K变异体代替Gly160Pro，具有大致相同的结果。
实施例75-78
漱口水组合物
实施例序号组份 75 76 77 78Ala166Gln+Ser216Asp 3.00 7.50 1.00 5.00SDA 40醇 8.00 8.00 8.00 8.00香料 0.08 0.08 0.08 0.08乳化剂 0.08 0.08 0.08 0.08氟化钠 0.05 0.05 0.05 0.05甘油 10.00 10.00 10.00 10.00甜味剂 0.02 0.02 0.02 0.02苯甲酸 0.05 0.05 0 05 0.05氢氧化钠 0.20 0.20 0.20 0.20染料 0.04 0.04 0.04 0.04水 平衡至100％
在实施例75-78中，用表2-36列出的其它蛋白酶K变异体代替Ala166Gln+Ser216Asp，具有大致相同的结果。
实施例79-82
锭剂组合物
实施例序号组份 79 80 81 82Gly70Ser+Ile107Gly+Leu133Met 0.01 0.03 0.10 0.02
+Phe192His+Asn194Asp山梨醇 17.50 17.50 17.50 17.50甘露醇 17.50 17.50 17.50 17.50淀粉 13.60 13.60 13.60 13.60甜味剂 1.20 1.20 1.20 1.20香料 11.70 11.70 11.70 11.70色素 0.10 0.10 0.10 0.10玉米糖浆 平衡至100％
在实施例79-82中，用表2-36列出的其它蛋白酶K变异体代替Gly70Ser+Ile107Gly+Leu133Met+Phe192His+Asn194Asp，具有大致相同的结果。
实施例83-86
口香糖组合物
实施例序号组份 83 84 85 86Gly214Asn 0.03 0.02 0.10 0.05山梨醇晶体 38.44 38.40 38.40 38.40Paloja-T胶基质* 20.00 20.00 20.00 20.00山梨醇(70％水溶液) 22.00 22.00 22.00 22.00甘露醇 10.00 10.00 10.00 10.00甘油 7.56 7.56 7.56 7.56香料 1.00 1.00 1.00 1.00*由L.A.Dreyfus公司提供。
在实施例83-86中，用表2-36列出的其它蛋白酶K变异体代替Gly214Asn，具有大致相同的结果。2.假牙清洗组合物
在本发明的另一个实施方案中，用于在口腔外洗涤假牙的假牙清洗组合物中含有本发明的一种或多种酶变异体。这些假牙清洗组合物含有有效量的一种或多种酶变异体，优选占组合物重量的大约0.0001％-50％，更优选占约0.001％-35％，最优选占约0.01％-20％，以及一种假牙洗涤载体。不同的假牙清洗组合物形式如起泡的药片等在本领域众所周知(参见Young的美国专利5,055,305，在此引入作为参考)，并通常适合掺入一种或多种酶变异体，以便从假牙上除去蛋白污垢。
本发明中假牙清洗组合物的实施方案在下面的实施例中详细说明。
实施例87-90
双层起泡假牙洗涤药片
实施例序号组份 87 88 89 90酸性层Tyr137Leu+Tyr169Cys+Phe192Asn 1.0 1.5 0.01 0.05+Ser207Asp+Arg218Glu+Ile220Leu酒石酸 24.0 24.0 24.00 24.00碳酸钠 4.0 4.0 4.00 4.00磺胺酸 10.0 10.0 10.00 10.00PEG 20,000 4.0 4.0 4.00 4.00碳酸氢钠 24.5 24.5 24.50 24.50过硫酸钾 15.0 15.0 15.00 15.00焦磷酸钠 7.0 7.0 7.00 7.00焦化硅石 2.0 2.0 2.00 2.00TAED* 7.0 7.0 7.00 7.00蓖麻酰磺化琥珀酸 0.5 0.5 0.50 0.50香料 1.0 1.0 1.00 1.00碱性层一水过硼酸钠 32.0 32.0 32.00 32.00碳酸氢钠 19.0 19.0 19.00 19.00EDTA 3.0 3.0 3.00 3.00三聚磷酸钠 12.0 12.0 12.00 12.00PEG 20,000 2.0 2.0 2.00 2.00过硫酸钾 26.0 26.0 26.00 26.00碳酸钠 2.0 2.0 2.00 2.00焦化硅石 2.0 2.0 2.00 2.00染料/香料 2.0 2.0 2.00 2.00*四乙酰乙二胺
在实施例87-90中，用表2-36列出的其它蛋白酶K变异体代替Tyr137Leu+Tyr169Cys+Phe192Ans+Ser207Asp+Arg218Glu-Ile220Leu，具有大致相同的结果。3.隐形眼镜清洗组合物
在本发明的另一个实施方案中，隐形眼镜清洗组合物包含一种或多种本发明的酶变异体。这些隐形眼镜清洗组合物中含有有效量的一种或多种酶变异体，优选大约占组合物重量的0.01％-50％，更优选约占0.01％-20％，最优选约占1％-5％，以及一种隐形眼镜洗涤载体。不同的隐形眼镜清洗组合物形式如药片、液体等在本领域中为人熟知(参见美国专利4,863,627,Davies，Meaken和Rees，1989年9月5日授权；美国专利4,609,493,Schfer，1986年9月2日授权；美国专利Re.32,672，Huth，Lam和Kirai，1988年5月24日授权；美国专利4,690,793,Ogunbiyi和Simth，1987年9月1日授权；美国专利4,614,549，Ogunbiyi，Riedhammer和Smith，1986年9月30日授权；和美国专利4,285,738，Ogata，1981年8月25日授权；所有这些在此引入作为参考)。并且通常适合掺入一种或多种本发明的酶变异体，以便从隐形眼镜上除去蛋白质性的污垢。
本发明中隐形眼镜清洗组合物的实施方案在下面的实施例中详细说明。
实施例91-94
加酶隐形眼镜洗涤溶液
实施例序号组份 91 92 93 94Gly102Gln 0.01 0.5 0.1 2.0葡萄糖 50.00 50.0 50.0 50.0非离子型表面活性剂(聚氧乙烯 2.00 2 0 2.0 2.0-聚氧丙烯共聚物)阴离子表面活性剂(聚氧乙烯- 1.00 1.0 1.0 1.0烷基苯醚硫酸钠)氯化钠 1.00 1.0 1.0 1.0硼砂 0.30 0.3 0.3 0.3水 平衡至100％
在实施例91-94中，用表2-36列出的其它蛋白酶K变异体代替Gly102Gln，具有大致相同的结果。
尽管本发明仅叙述了具体实施方案，但很显然对本领域的技术人员来说，可以在不超出本发明的精神和范围的情况下对本发明作各种改变和修改。本发明意在后附的权利要求中包括在本发明范围之内的所有这些修改。
序列表(1)一般信息
(i)申请人：BRODE，PHILIP F
BARNETT，BOBBY L
RUBINGH，DONN N
(ii)发明题目：能降低吸附作用并提高水解作用的蛋白酶K变异
体
(iii)序列数：1
(iv)通讯地址：
(A)收件人：THE PROCTER & GAMBLE COMPANY
(B)街道：11810 EAST MIAMIRIVER ROAD
(C)城市：ROSS
(D)州：OH
(E)国家：US
(F)邮编：45061
(v)计算机可读形式：
(A)介质类型：软盘
(B)计算机：IBM PC兼容
(C)操作系统：PC-DOS/MS-DOS
(D)软件：PatentIn Release#1.0，Version#1.30
(vi)现申请资料：
(A)申请号：
(B)申请日：
(C)分类：
(viii)代理人/事务所资料：
(A)姓名：HAKE，RICHARD A
(B)注册号：37，343
(C)参考/摘要号：5605
(ix)电信信息：
(A)电话：(513)-627-0087
(B)传真：(513)-627-0260(2)SEQ ID NO：1信息
(i)序列特征
(A)长度：279氨基酸
(B)类型：氨基酸
(C)链型：单链
(D)拓扑学：线性
(xi)序列表述：SEQ ID NO：1：Ala Ala Gln Thr Asn Ala Pro Trp Gly Leu Ala Arg Ile Ser Ser Thr1 5 10 15Ser Pro Gly Thr Ser Thr Tyr Tyr Tyr Asp Glu Ser Ala Gly Gln Gly
20 25 30Ser Cys Val Tyr Val Ile Asp Thr Gly Ile Glu Ala Ser His Pro Glu
35 40 45Phe Glu Gly Arg Ala Gln Met Val Lys Thr Tyr Tyr Tyr ser Ser Arg
50 55 60Asp Gly Asn Gly His Gly Thr His Cys Ala Gly Thr Val Gly Ser Arg65 70 75 80Thr Tyr Gly Val Ala Lys Lys Thr Gln Leu Phe Gly Val Lys Val Leu
85 90 95Asp Asp Asn Gly Ser Gly Gln Tyr Ser Thr Ile Ile Ala Gly Mer Asp
100 105 110Phe Val Ala Ser Asp Lys Asn Asn Arg Asn Cys Pro Lys Gly Val Val
115 120 125Ala Ser Leu Ser Leu Gly Gly Gly Tyr Ser Ser Ser Val Asn Ser Ala
130 135 140Ala Ala Arg Leu Gln Ser Ser Gly Val Met Val Ala Val Ala Ala Gly145 150 155 160Asn Asn Asn Ala Asp Ala Arg Asn Tyr Ser Pro Ala Ser Glu Pro Ser
165 170 175Val Cys Thr Val Gly Ala Ser Asp Arg Tyr Asp Arg Arg Ser Ser Phe
180 185 190Ser Asn Tyr Gly Ser Val Leu Asp Ile Phe Gly Pro Gly Thr Ser Ile
195 200 205Leu Ser Thr Trp Ile Gly Gly Ser Thr Arg Ser Ile Ser Gly Thr Ser
210 215 220Met Ala Thr Pro His Val Ala Gly Leu Ala Ala Tyr Leu Met Thr Leu225 230 235 240Gly Lys Thr Thr Ala Ala Ser Ala Cys Arg Tyr Ile Ala Asp Thr Ala
245 250 255Asn Lys Gly Asp Leu Ser Asn Ile Pro Phe Gly Thr Val Asn Leu Leu
260 265 270Ala Tyr Asn Asn Tyr Gln Ala
275
Claims (9)
1.一种对野生型蛋白酶K的氨基酸序列进行改良的蛋白酶K变异体，野生型氨基酸序列中包含第一环区、第二环区、第三环区、第四环区、第五环区和第六环区，其中改良的氨基酸序列在一个或多个环区中的一个或多个位置上有替换；其中：
A.当替换发生在第一环区时，替换发生在64、65、66、68、
70或71位中的一个或多个上；其中
a.当替换发生在64位时，替代氨基酸为Asp或Glu；
b.当替换发生在65位时，替代氨基酸为Glu；
c.当替换发生在66位时，替代氨基酸为Asn、Asp、Gln、
Glu、Pro或Ser；
d.当替换发生在68位时，替代氨基酸为Asn、Asp、Gln、
Glu、Pro或Ser；
e.当替换发生在70位时，替代氨基酸为Asn、Asp、Gln、
Glu、Pro或Ser；
f.当替换发生在71位时，替代氨基酸为Asn、Asp、Gln、
Glu、Pro或Ser；
B.当替换发生在第二环区时，替代发生在95、96、97、98、
100、102、103、104、106或107位中一个或多个上；其中
a.当替换发生在95位时，替代氨基酸为Ala、Asn、Asp、
Cys、Gln、Glu、Gly、His、Met、Pro、Ser或Thr；
b.当替换发生在96位时，替代氨基酸为Ala、Asn、Asp、
Cys、Gln、Glu、Gly、His、Ile、Met、Pro、Ser、
Thr或Val；
c.当替换发生在97位时，替代氨基酸为Glu；
d.当替换发生在98位时，替代氨基酸为Glu；
e.当替换发生在100位时，替代氨基酸为Asn、Gln、Pro或
Ser；
f.当替换发生在102位时，替代氨基酸为Asn、Gln、Glu、
Pro或Ser；
g.当替换发生在103位时，替代氨基酸为Asn或Ser；
h.当替换发生在104位时，替代氨基酸为Asp、His、Ile、
Leu、Met、Pro或Val；
i.当替换发生在106位时，替代氨基酸为Asn、Gln、Gly、
Pro或Ser；和
j.当替换发生在107位时，替代氨基酸为Ala、Asn、Cys、
Gln、Gly、His、Leu、Met、Pro、Ser、Thr或Val；
C.当替换发生在第三环区时，替代发生在133、134、136、137、
138、139或140位中一个或多个上；其中
a.当替换发生在133位时，替代氨基酸为Ala、Asn、Cys、
Gln、Gly、His、Met、Pro、Ser、Thr或Val；
b.当替换发生在134位时，替代氨基酸为Asn、Asp、Gln、
Glu、Pro或Ser；
c.当替换发生在136位时，替代氨基酸为Asn、Asp、Gln、
Glu、Pro或Ser；
d.当替换发生在137位时，替代氨基酸为Asp、His、Ile、
Leu、Met或Pro；
e.当替换发生在138位时，替代氨基酸为Asp或Glu；
f.当替换发生在139位时，替代氨基酸为Asp或Glu；和
g.当替换发生在140位时，替代氨基酸为Asp或Glu；
D.当替换发生在第四环区时，替换发生在161、162、163、164、
165、166、167、168、169或170位中一个或多个上；其中
a.当替换发生在161位时，替代氨基酸为Gln；
b.当替换发生在162位时，替代氨基酸为Asp、Gln、Glu或
Ser；
c.当替换发生在163位时，替代氨基酸为Asp、Gln、Glu或
Ser；
d.当替换发生在164位时，替代氨基酸为Asn、Asp、Gln、
Glu、Gly、His、Pro、Ser或Thr；
e.当替换发生在165位时，替代氨基酸为Glu；
f.当替换发生在166位时，替代氨基酸为Asn、Asp、Gln、
Glu、Gly、His、Pro、Ser或Thr；
g.当替换发生在167位时，替代氨基酸为Asp或Glu；
h.当替换发生在168位时，替代氨基酸为Asp、Gln、Glu或
Ser；
i.当替换发生在169位时，替代氨基酸为Asp、His、Ile、
Leu、Met或Pro；和
j.当替换发生在170位时，替代氨基酸为Asp或Glu；
E.当替换发生在第五环区时，替换发生在190、192、193或194
位中的一个或多个上，其中
a.当替换发生在190位时，替代氨基酸为Asp或Glu；
b.当替换发生在192位时，替代氨基酸为Asn、Cys、Gln、
His、Ile、Met、Pro、Thr、Tyr或Val；
c.当替换发生在193位时，替代氨基酸为Asp或Glu；和
d.当替换发生在194位时，替代氨基酸为Asp、Gln、Glu或
Ser；和
F.当替换发生在第六环区时，替换发生在203、204、205、206、
208、209、210、211、212、213、214、215、216、217、
218、219、220、221、222或223位中的一个或多个上；其
中
a.当替换发生在203位时，替代氨基酸为Asn、Asp、Gln、
Glu、Pro或Ser；
b.当替换发生在204位时，替代氨基酸为Asn、Asp、Gln、
Glu、Gly或Ser；
c.当替换发生在205位时，替代氨基酸为Asn、Asp、Gln、
Glu、Pro或Ser；
d.当替换发生在206位时，替代氨基酸为Asn、Asp、Gln、
Glu、Gly、Pro或Ser；
e.当替换发生在208位时，替代氨基酸为Ala、Asn、Asp、
Cys、Gln、Glu、Gly、His、Leu、Met、Pro、Ser、
Thr或Val；
f.当替换发生在209位时，替代氨基酸为Ala、Asn、Asp、
Cys、Gln、Glu、Gly、His、Ile、Met、Pro、Ser、
Thr或Val；
g.当替换发生在210位时，替代氨基酸为Asp或Glu；
h.当替换发生在211位时，替代氨基酸为Asn、Asp、Gln、
Glu、Pro或Ser；
i.当替换发生在212位时，替代氨基酸为Asp或Glu；
j.当替换发生在213位时，替代氨基酸为Ala、Asn、Asp、
Cys、Gln、Glu、Gly、His、Leu、Met、Pro、Ser、
Thr或Val；
k.当替换发生在214位时，替代氨基酸为Asn、Gln、Pro或
Ser；
l.当替换发生在215位时，替代氨基酸为Asn、Asp、Gln、
Glu、Pro或Ser；
m.当替换发生在216位时，替代氨基酸为Asp或Glu；
n.当替换发生在217位时，替代氨基酸为Asn、Asp、Gln、
Glu、Gly、Pro或Ser；
o.当替换发生在218位时，替代氨基酸为Asp或Glu；
p.当替换发生在219位时，替代氨基酸为Asp或Glu；
q.当替换发生在220位时，替代氨基酸为Ala、Asn、Asp、
Cys、Gln、Glu、Gly、His、Leu、Met、Pro、Ser、
Thr或Val；
r.当替换发生在221位时，替代氨基酸为Asp或Glu；
s.当替换发生在222位时，替代氨基酸为Asn、Asp、Gln、
Glu、Pro或Ser；
t.当替换发生在223位时，替代氨基酸为Asn、Asp、Gln、
Glu、Pro或Ser；因此与野生型蛋白酶K相比，蛋白酶K变异体降低了对不溶底物的吸附作用而提高了对不溶底物的水解作用。
2.权利要求1的蛋白酶K变异体，其中在第一环区发生了一个或多个替换。
3.权利要求1的蛋白酶K变异体，其中在第二环区发生了一个或多个替换。
4.权利要求1的蛋白酶K变异体，其中在第三环区发生了一个或多个替换。
5.权利要求1的蛋白酶K变异体，其中在第四环区发生了一个或多个替换。
6.权利要求1的蛋白酶K变异体，其中在第五环区发生了一个或多个替换。
7.权利要求1的蛋白酶K变异体，其中在第六环区发生了一个或多个替换。
8.一种选自硬表面洗涤组合物、餐具洗涤组合物、口腔清洁组合物、假牙清洗组合物、隐形眼镜清洗组合物以及织物洗涤组合物的清洗组合物，其特征在于该清洗组合物含有权利要求1-7中任一项中的蛋白酶K变异体以及一种洗涤组合物载体；优选洗涤组合物为硬表面洗涤组合物或织物洗涤组合物；优选该组合物至少含有占总重约5％的表面活性剂和至少约5％的助洗剂；优选该组合物还包含选自以下的洗涤组合物成份：溶剂、缓冲剂、酶、污垢解脱剂、粘土类去污剂、分解剂、增白剂、抑泡剂、织物柔软剂、发泡剂、酶稳定剂、漂白剂、染料、香料及其混合物。
9.编码权利要求1-7中任一项中的蛋白酶K变异体的DNA序列。
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US40157495A | 1995-03-09 | 1995-03-09 | |
| US08/401,574 | 1995-03-09 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1183809A true CN1183809A (zh) | 1998-06-03 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN96193792A Pending CN1183809A (zh) | 1995-03-09 | 1996-03-06 | 具有降低吸附作用提高水解作用的蛋白酶k变异体 |
Country Status (14)
| Country | Link |
|---|---|
| US (1) | US6451574B1 (zh) |
| EP (1) | EP0813601A2 (zh) |
| JP (1) | JPH11501814A (zh) |
| CN (1) | CN1183809A (zh) |
| AU (1) | AU5092796A (zh) |
| BR (1) | BR9607677A (zh) |
| CA (1) | CA2214395C (zh) |
| CZ (1) | CZ279997A3 (zh) |
| HU (1) | HUP9801638A3 (zh) |
| IL (1) | IL117350A0 (zh) |
| MA (1) | MA23820A1 (zh) |
| MX (1) | MX9706841A (zh) |
| WO (1) | WO1996028556A2 (zh) |
| ZA (1) | ZA961820B (zh) |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105193640A (zh) * | 2014-06-24 | 2015-12-30 | 金普诺安蛋白质工程技术（北京）有限公司 | 蛋白酶k在皮肤保健和化妆品领域中的应用 |
| CN113913412A (zh) * | 2021-10-13 | 2022-01-11 | 湖北大学 | 蛋白酶k突变体及其制备方法 |
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- 1996-03-06 EP EP96907182A patent/EP0813601A2/en not_active Withdrawn
- 1996-03-06 CN CN96193792A patent/CN1183809A/zh active Pending
- 1996-03-06 BR BR9607677A patent/BR9607677A/pt not_active Application Discontinuation
- 1996-03-06 JP JP8527690A patent/JPH11501814A/ja active Pending
- 1996-03-06 HU HU9801638A patent/HUP9801638A3/hu unknown
- 1996-03-06 CZ CZ972799A patent/CZ279997A3/cs unknown
- 1996-03-06 ZA ZA961820A patent/ZA961820B/xx unknown
- 1996-03-06 AU AU50927/96A patent/AU5092796A/en not_active Abandoned
- 1996-03-06 WO PCT/US1996/003005 patent/WO1996028556A2/en not_active Application Discontinuation
- 1996-03-06 CA CA002214395A patent/CA2214395C/en not_active Expired - Fee Related
- 1996-03-07 MA MA24177A patent/MA23820A1/fr unknown
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1997
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105193640A (zh) * | 2014-06-24 | 2015-12-30 | 金普诺安蛋白质工程技术（北京）有限公司 | 蛋白酶k在皮肤保健和化妆品领域中的应用 |
| CN105193640B (zh) * | 2014-06-24 | 2018-10-12 | 金普诺安蛋白质工程技术（北京）有限公司 | 蛋白酶k在皮肤保健和化妆品领域中的应用 |
| CN113913412A (zh) * | 2021-10-13 | 2022-01-11 | 湖北大学 | 蛋白酶k突变体及其制备方法 |
| CN113913412B (zh) * | 2021-10-13 | 2023-10-03 | 湖北大学 | 蛋白酶k突变体及其制备方法 |
Also Published As
| Publication number | Publication date |
|---|---|
| BR9607677A (pt) | 1998-07-07 |
| WO1996028556A2 (en) | 1996-09-19 |
| CA2214395C (en) | 2001-10-30 |
| MX9706841A (es) | 1997-11-29 |
| IL117350A0 (en) | 1996-07-23 |
| EP0813601A2 (en) | 1997-12-29 |
| CA2214395A1 (en) | 1996-09-19 |
| MA23820A1 (fr) | 1996-10-01 |
| AU5092796A (en) | 1996-10-02 |
| ZA961820B (en) | 1996-09-10 |
| CZ279997A3 (cs) | 1998-01-14 |
| US6451574B1 (en) | 2002-09-17 |
| HUP9801638A2 (hu) | 1998-10-28 |
| HUP9801638A3 (en) | 2001-01-29 |
| WO1996028556A3 (en) | 1997-07-17 |
| JPH11501814A (ja) | 1999-02-16 |
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