CN111183220B - 用于聚糖分析的工具 - Google Patents

用于聚糖分析的工具 Download PDF

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CN111183220B
CN111183220B CN201880050240.9A CN201880050240A CN111183220B CN 111183220 B CN111183220 B CN 111183220B CN 201880050240 A CN201880050240 A CN 201880050240A CN 111183220 B CN111183220 B CN 111183220B
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弗雷德里克·里奥
罗尔夫·卢德
史蒂芬·比约克
马林·梅贾尔
弗雷德里克·奥尔森
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    • C12N9/24Hydrolases (3) acting on glycosyl compounds (3.2)
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    • C12N9/14Hydrolases (3)
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    • C12N9/52Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
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    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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    • G01N2333/95Proteinases, i.e. endopeptidases (3.4.21-3.4.99)
    • G01N2333/952Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from bacteria

Abstract

本发明涉及用于研究糖蛋白的酶及其组合,以及相应的使用方法。特别地,本发明涉及包含另外的蛋白酶和/或糖苷酶的唾液酸酶组合物,该另外的蛋白酶和/或糖苷酶优选O‑糖蛋白特异性内切蛋白酶和/或O‑糖苷酶。

Description

用于聚糖分析的工具
技术领域
本发明涉及用于研究糖蛋白的酶及其组合,以及相应的使用方法。
背景技术
蛋白质的糖基化在人的许多生理功能中起着关键作用,包括信号传导、运输、防止蛋白水解活性、粘附、炎症反应、微生物定植等。大多数与蛋白质连接的聚糖链,无论是O-连接还是N-连接的,都修饰有末端唾液酸。作为糖蛋白上最外层的聚糖,它们的存在/缺乏通常对下游效应(包括例如免疫球蛋白的炎症潜能)至关重要。就在给定蛋白质上的存在/缺乏以及单个结构修饰而言,蛋白质上的唾液酸是异源的。它们通常还带有负电荷,使质谱分析复杂化。这使得对糖蛋白的研究变得困难,并且降低了制造商确认糖蛋白批次将在同源物质中起作用的能力。为了克服这些问题,已经尝试对CHO细胞进行基因工程以降低聚糖的复杂性,尽管这可能影响功能。也已经使用化学方法,但是这些通常会破坏蛋白质。需要从糖蛋白去除唾液酸的可供选择的方法。此外,一旦去除了唾液酸,就需要更多的工具来研究剩余的聚糖链,特别是那些O-连接的聚糖链。
发明内容
本发明提供:
一种组合物,所述组合物包含第一唾液酸酶,第一唾液酸酶独立地选自:
(a)包含以下或由以下组成的多肽:SEQ ID NO:2氨基酸序列;
(b)包含以下或由以下组成的多肽:与SEQ ID NO:2氨基酸序列至少85%一致的氨基酸序列;或
(c)包含以下氨基酸序列或由以下氨基酸序列组成的多肽:该氨基酸序列为SEQID NO:2序列的片段或与SEQ ID NO:2氨基酸序列85%一致的氨基酸的片段;
任选地,其中所述第一唾液酸酶在N末端包括另外的甲硫氨酸和/或在C末端包括组氨酸标签,该标签可以通过接头与C末端连接。
本发明还提供了一种如上定义的组合物,该组合物还包含第二唾液酸酶,所述第二唾液酸酶独立地选自:
(d)包含以下或由以下组成的多肽:SEQ ID NO:5氨基酸序列;
(e)包含以下或由以下组成的多肽:与SEQ ID NO:5氨基酸序列至少85%一致的氨基酸序列;或
(f)包含以下氨基酸序列或由以下氨基酸序列组成的多肽:该氨基酸序列为SEQID NO:5序列的片段或与SEQ ID NO:5氨基酸序列85%一致的氨基酸的片段。
任选地,其中所述第二唾液酸酶在N末端包括另外的甲硫氨酸和/或在C末端包括组氨酸标签,该标签可以通过接头与C末端连接。
该组合物可以另外包含糖苷酶和/或蛋白酶,所述糖苷酶和/或蛋白酶任选地以高度纯化或分离的形式存在。
本发明还提供了一种用于修饰糖蛋白的方法,该方法包括使包含糖蛋白的样品与如上所定义的组合物接触,任选地其中对所得产物进行分析。
附图说明
图1:艾克曼菌(Akkermansia)唾液酸酶的表达和纯化。本文中以Am0707和Am1757为代表的所有四种唾液酸酶均表达良好,并且能够纯化至高度均一。值(mg/mL)表示His纯化后获得的浓度。
图2:在不同唾液酸键情况下的唾液酸酶活性。将两种唾液酸酶产物(Am1757和Mix(Am1757和Am0707))与三种不同的底物(代表3种指示的唾液酸键类型)孵育30分钟,然后测量游离唾液酸的量。
图3:确定Am0707和Am1757的最佳条件。使用2至3种唾液乳糖底物,通过孵育15分钟后对游离唾液酸进行定量,来研究影响唾液酸酶的酶促活性的条件,包括离子、NaCl和pH。
图4:用组合唾液酸酶处理糖蛋白比使用单种唾液酸酶更有效。将唾液酸酶与0.5μg胎球蛋白孵育60分钟,然后使用SDS-PAGE将它们分开。Smix同时包含Am0707和Am1757,而使用产气荚膜梭菌(C.perfringens)唾液酸酶作为基准对照。除根据制造商说明进行孵育的基准产品外,所有反应均在37℃下在20mM Tris-HCl pH 6.8中进行。
图5:GVS_Smix优于目前可商购的唾液酸酶。来自New England Biolabs的数种现有商业唾液酸酶(每组中的前3列)与AM1757+Am0707混合物(GVS_Smix)以及Am1757和Am0707中的每种酶分别并行测试。在分析之前,各自与特定唾液酸酶底物(代表3种指示的唾液酸键类型)在37℃下孵育30分钟。FU(荧光单位)代表释放的唾液酸的量。
图6:GVS_Smix可以使胎球蛋白完全去唾液酸化。SDS-PAGE和SNA印迹显示Smix(AM1757+Am0707混合物)以及两种New England Biolabs产品(NEB A和NEB O)可以使胎球蛋白中的2-3和2-6唾液酸键完全去唾液酸化。两种2-3特异性酶Am1757和New EnglandBiolabs NEB S无法使胎球蛋白完全去唾液酸化。
图7:GVS_Smix(AM1757+Am0707混合物;第一组柱)以与所有三种测试的商业化产品(后三组柱)相似或更好的水平从天然蛋白质中释放唾液酸。还显示了单独的Am1757酶(第二组柱)。蛋白质与唾液酸酶在其各自缓冲液中混合,并孵育15分钟,然后添加唾液酸显影缓冲液。所有孵育均在37℃下在各自的缓冲液中进行(对于GVS-Smix,缓冲液是20mMTris-HCl pH 6.8)。
图8:O-糖苷酶的重组表达。(A)表达来自口腔链球菌(S.oralis)和两歧双歧杆菌(B.bifidum)的O-糖苷酶,亲和纯化并在SDS-PAGE上分开。(B)在4℃下储存0至6天的口腔链球菌O-糖苷酶的稳定性测定。
图9:O-糖苷酶可以水解Core 1、2和3型pNP标记的O-糖苷。将来自口腔链球菌(S.o)和两歧双歧杆菌(B.b)的不同浓度的O-糖苷酶与(A)Core 1、(B)Core 2和(C)Core 3O-聚糖孵育,根据405nm吸光度变化测量pNP释放。
图10:口腔链球菌糖苷酶在补充有MgCl2的碱性pH中具有较高的活性。在不同pH下(A),以不同离子(B)和不同浓度的MgCl2(C)孵育O-糖苷酶以确定最佳缓冲液。
图11:O-糖苷酶活性是时间和剂量依赖性的。将TNFαR(1μg)与仅GVS_Smix唾液酸酶混合物(标有TNFaR(Smix)的泳道)孵育作为对照,或与GVS_Smix唾液酸酶混合物和示出的来自口腔链球菌(S.o)的不同量的O-糖苷酶的组合(“+5ug So等”)孵育。所指示的添加的O-糖苷酶的量依赖于nanodrop的值。然而,由于片段化,全长蛋白质的实际添加量接近书面值的约10%至20%。在补充有2mM CaCl2但无MgCl2的20mM Tris-HCl pH 8.0中孵育这些样品。
图12:O-糖苷酶可以作用于研究的所有天然糖蛋白。将不同的天然蛋白质(P)与唾液酸酶(Smix)和来自口腔链球菌(So-glyk)和双歧双歧杆菌(Bb-glyk)的O-糖苷酶的GVS_Smix混合物孵育。过夜孵育,然后在SDS-PAGE上分析产物。
图13:对于抗天然糖蛋白的作用,口腔链球菌O-糖苷酶比可商购的产品有利。将天然TNFαR与仅GVS_Smix唾液酸酶混合物(标有TNFaR(Smix)的泳道)孵育作为对照,或与GVS_Smix唾液酸酶混合物和来自口腔链球菌(S.o)的示出的不同量的O-糖苷酶的组合(“+So(0.5ug)”等)孵育,或与所示量的可商购的O-糖苷酶(NEB)制造商随该可商购的O-糖苷酶(NEB)一起提供的唾液酸酶(“+NEB(3ul)”等)孵育。将所有酶与TNFαR在其各自的缓冲液中孵育1小时或16小时。口服链球菌糖苷酶的最高剂量(“So(0.5ug)”)在摩尔浓度上大约等于0.3ul商业O-糖苷酶(NEB)。NEB处理的样品中,明显的低分子量(凝胶顶部)条带是唾液酸酶。
图14:聚糖组成影响活性。在添加O-糖苷酶(So)之前,用唾液酸酶(S)和/或半乳糖苷酶(G)孵育底物;在最佳缓冲液(20mM Tris-HCl pH 8.0)中于37℃下过夜孵育。
图15:最终产品是具有竞争力的。使用酶:底物比例为1:40的O-糖苷酶,以及唾液酸酶混合物中的酶底物比例为1:40+1:40的Am1757+Am0707(所有这三种酶均在20mM Tris-HCl pH 6.8中(标记为Smix/O-glyk的泳道)),将口腔链球菌O-糖苷酶的活性与可商购的O-糖苷酶+唾液酸酶组合的活性(标记为NEB(O-glyk试剂盒)的泳道)进行比较。使用ConA(N-连接的聚糖)和木菠萝素(O-连接的聚糖),用凝集素印迹验证通过口腔链球菌O-糖苷酶+唾液酸酶混合物(“+GVS”)从TNFaR去除O-聚糖,未处理的TNFaR作为对照。
图16:O-糖苷酶+唾液酸酶束(Smix/O-glyk)对多种糖蛋白具有活性。将以终浓度添加的酶和在20mM Tris-HCl pH 6.8中的制剂(1:40)在37℃下孵育4小时,然后在SDS-PAGE上分开。
图17:特定的唾液酸键影响O-糖苷酶活性。将以终浓度添加的酶和在20mM Tris-HCl pH6.8中的制剂(1:40)与TNFαR在37℃下孵育15分钟至4小时,然后在SDS-PAGE上分开。
图18:口腔链球菌O-糖苷酶至少部分地由于有效的唾液酸酶而突出。将口腔链球菌O-糖苷酶(GVS O-glyk)或商业O-糖苷酶(NEB O-glyk)与GVS_Smix唾液酸酶混合物(GVS唾液酸酶)孵育或与商业唾液酸酶(NEB A、NEB S、NEB O)孵育。使用适当的缓冲液(例如,对于GVS,使用20mM Tris-HCl pH 6.8),将每组酶与Enbrel(依那西普)在37℃下孵育4小时,然后在SDS-PAGE上使产物分开。
图19:2-3键合的唾液酸限制了O-糖蛋白特异性内切蛋白酶(LS)的效率。使用Enbrel作为糖蛋白底物,将LS与一组多样化的唾液酸酶同时孵育30分钟至20小时,揭示了在存在2-3特异性唾液酸酶1757的情况下或在Mix(0707+1757)的情况下具有更高效率,而广谱唾液酸酶0707对于LS的近似完全活性不是必需的,因此表明2-6(和2-8)键与LS活性无关。
序列的简要说明
SEQ ID NO:1、2和3分别是从嗜粘蛋白艾克曼菌(Akkermansia muciniphila)分离出的唾液酸酶的氨基酸序列。SEQ ID NO:1是在N末端包括信号基序的野生型序列。SEQ IDNO:2是去除了信号基序的野生型序列。SEQ ID NO:3与SEQ ID NO:2一致,除了SEQ ID NO:3包括另外的N末端甲硫氨酸和C末端接头+His6标签。包含SEQ ID NO:2序列的任何序列(包括SEQ ID NO:1至3中的每个)在本文中可以称为Am0707。
SEQ ID NO:4、5和6分别是从嗜粘蛋白艾克曼菌分离出的另一种唾液酸酶的氨基酸序列。SEQ ID NO:4是在N末端包括信号基序的野生型序列。SEQ ID NO:5是去除了信号基序的野生型序列。SEQ ID NO:6与SEQ ID NO:5一致,除了SEQ ID NO:6包括另外的N末端甲硫氨酸和C末端接头+His6标签。包含SEQ ID NO:5序列的任何序列(包括SEQ ID NO:4至6中的每个)在本文中可以称为Am1757。
SEQ ID NO:7、8、9和10分别是从口腔链球菌分离出的O-糖苷酶的氨基酸序列。SEQID NO:7是在N末端包含信号基序并且在C末端包含LPXTG壁锚定基序的野生型序列。SEQ IDNO:8是去除了信号基序的野生型序列。SEQ ID NO:9是去除了信号基序和壁锚定基序的野生型序列。SEQ ID NO:10与SEQ ID NO:9一致,除了SEQ ID NO:10包括另外的N末端甲硫氨酸和C末端接头+His6标签。包含SEQ ID NO:9序列的任何序列(包括SEQ ID NO:7至10中的每个)在本文中可以被称为“O-glyk”或“So”。
SEQ ID NO:11是O-糖蛋白特异性内切蛋白酶的氨基酸序列。
SEQ ID NO:12是具有O-糖蛋白特异性内切蛋白酶活性的示例性多肽的氨基酸序列。相对于SEQ ID NO:11,SEQ ID NO:12包括另外的N末端甲硫氨酸和C末端接头+His6标签。由该序列组成的多肽在本文中可以称为LS。
SEQ ID NO:13是编码由SEQ ID NO:10氨基酸序列组成的多肽的示例性核酸序列。
具体实施方式
应当理解,所公开的产品和方法的不同应用可以适合于本领域的特定需求。还应理解,本文所用的术语仅出于描述本发明的特定实施方式的目的,而并非旨在进行限制。本文引用的所有出版物、专利和专利申请,无论是上文还是下文中的,均通过引用全文并入本文。
除非文中另外明确指出,如在本说明书和所附权利要求书中所使用的,单数形式“一个/一种(a/an)”和“该(the)”包括复数个指示物。因此,例如,提及“多肽(apolypeptide)”包括“多个多肽(polypeptides)”等。
本说明书特别涉及多肽,该多肽为唾液酸酶、O-糖苷酶和O-糖蛋白特异性内切蛋白酶。因此,术语多肽的一般用途可以应用于这些类型的酶中的每一种中。
一般的多肽特征
“多肽”在本文以最广泛的意义使用,是指两个或更多个亚基氨基酸、氨基酸类似物或其他拟肽的化合物。因此,术语“多肽”包括短肽序列以及更长的多肽和蛋白质。术语“蛋白质”、“肽”和“多肽”可以互换使用。如本文所用,术语“氨基酸”是指天然和/或非天然或合成的氨基酸,包括D或L旋光异构体,以及氨基酸类似物和拟肽。
多肽可以通过合适的方法产生,包括重组或合成方法。例如,多肽可以使用本领域已知的标准技术直接合成,例如Fmoc固相化学、Boc固相化学或通过溶液相肽合成。可供选择地,可以通过用编码所述多肽的核酸分子或载体转化细胞,通常转化细菌细胞来产生多肽。下文描述了并且在实施例中举例说明了通过在细菌宿主细胞中表达来产生多肽的方法。本发明提供了编码本发明多肽的核酸分子和载体。本发明还提供了包含这种核酸或载体的宿主细胞。编码本文公开的多肽的示例性多核苷酸分子以SEQ ID NO:13提供。该序列在3'端包括N末端甲硫氨酸(ATG)的密码子,并且在5'端在终止密码子(TAA)之前包括GSGLE接头和6x His标签的密码子,可以任选不包括所述GSGLE接头和6x His标签的密码子。下文将更详细地讨论另外的甲硫氨酸和标签的任性包含。
术语“核酸分子”和“多核苷酸”在本文可互换使用,并且是指任何长度的核苷酸(无论是脱氧核糖核苷酸还是核糖核苷酸)的聚合形式或其类似物。多核苷酸的非限制性实例包括基因、基因片段、信使RNA(mRNA)、cDNA、重组多核苷酸、质粒、载体、任何序列的分离的DNA、任何序列的分离的RNA、核酸探针和引物。本发明的多核苷酸编码本发明的多肽,并且可以以分离的或基本上分离的形式提供。基本上分离是指可以从任何周围介质中基本上但不完全分离多肽。可以将多核苷酸与不会干扰其预期用途的运载体或稀释剂混合,并且仍然被认为是基本上分离的。“编码”所选多肽的核酸序列是这样一种核酸分子,所述核酸分子在受适当调控序列控制时,在活体内转录(对于DNA而言)并翻译(对于mRNA而言)成多肽,例如在表达载体中。编码序列的边界由5'(氨基)末端的起始密码子和3'(羧基)末端的翻译终止密码子确定。出于本发明的目的,这样的核酸序列可以包括但不限于来自病毒的cDNA、原核或真核mRNA、来自病毒的基因组序列、或原核DNA或RNA,甚至合成的DNA序列。转录终止序列可以位于编码序列的3'处。
多核苷酸可以根据本领域众所周知的方法来合成,如例如Sambrook et al.(1989,分子克隆-实验指南(Molecular Cloning-a laboratory manual);冷泉港出版社)中所描述。本发明的核酸分子可以以表达盒的形式提供,所述表达盒包括可操作地连接至插入序列的控制序列,从而允许本发明的多肽在活体内表达。这些表达盒又通常在载体(例如质粒或重组病毒载体)内提供。可以将这样的表达盒直接给药至宿主对象。可供选择地,可以将包含本发明的多核苷酸的载体给药至宿主对象。优选地,使用遗传载体进行多核苷酸制备和/或给药。合适的载体可以是能够携带足量的遗传信息并允许表达本发明的多肽的任何载体。
因此,本发明包括包含这样的多核苷酸序列的表达载体。这样的表达载体在分子生物学领域中是常规构建的,并且例如可以涉及使用质粒DNA和适当的起始子、启动子、增强子和其他元件,例如,为了允许表达本发明的肽而可能是必要的并且以正确的取向定位的聚腺苷酸化信号。其他合适的载体对本领域技术人员来说将是显而易见的。作为在这方面的进一步的实例,我们参考Sambrook et al.。
本发明还包括已经修饰以表达本发明的多肽的细胞。这样的细胞通常包括原核细胞,例如细菌细胞,例如大肠杆菌。可以使用常规方法培养这样的细胞以产生本发明的多肽。
多肽可以衍生化或经修饰以有助于该多肽的产生、分离或纯化。例如,在通过细菌宿主细胞中的重组表达来产生本发明的多肽的情况下,该多肽的序列可以在N末端包含另外的甲硫氨酸(M)残基以改善表达。作为另一个实例,本发明的多肽可以通过添加能够直接且特异性地与分开手段结合的配体来衍生化或进行修饰。可供选择地,多肽可以通过添加结合对中的一个成员来衍生化或进行修饰,并且分开手段包括通过添加结合对中的其他成员而衍生化或进行修饰的试剂。可以使用任何合适的结合对。在用于本发明的多肽通过添加结合对中的一个成员而衍生化或进行修饰的优选实施方式中,该多肽优选是组氨酸标记的或生物素标记的。通常,在基因水平上包括组氨酸或生物素标签的氨基酸编码序列,并且该多肽在大肠杆菌中进行重组表达。组氨酸或生物素标签通常存在于多肽的任一端,优选地在C末端。组氨酸或生物素标签可以与多肽直接连接或通过任何合适的接头序列(例如3、4或5个氨基酸)间接连接。接头通常可以主要由甘氨酸和丝氨酸残基组成。组氨酸标签通常由六个组氨酸残基组成,尽管组氨酸标签可以比这更长,通常多达7、8、9、10或20个氨基酸,或更短,例如5、4、3、2或1个氨基酸。
多肽可以以基本上分离的或纯化的形式提供。也就是说,从存在于表达多肽的细胞的细胞提取物中的其他大部分成分中分离的。将理解的是,基本上纯化是指多肽被纯化至至少50%、60%、70%、80%或优选至少90%同质性。纯度水平可通过任何合适的方法进行评估,但通常涉及样品的SDS-PAGE分析,然后进行考马斯蓝检测。多肽可以与不会干扰多肽预期目的的运载体、稀释剂或防腐剂混合,并且仍被为是基本上分离的或纯化的。在多肽提供于具有另外的活性成分例如另一种多肽的组合物中的情况下,则将每种所述多肽分别纯化至高水平的同质性,然后以适当的比例混合以用于各自的预期目的。例如,可以将两种多肽各自纯化至至少90%的同质性,然后以1:1的比例组合。
多肽(或其混合物)可以以适于在使用前在水溶液中重构的冻干形式提供。冻干的组合物具有改善的稳定性,使得能够更长久地储存多肽。本文提供了一种制备冻干形式的多肽(或其混合物)的方法,所述方法包括在合适的缓冲液(例如Tris-缓冲盐水(TBS))中冷冻干燥所述多肽(或混合物)。多肽在冷冻干燥之前通常是基本上纯化的。还提供了冻干形式的所得多肽(或混合物)。还提供了一种制备多肽(或混合物)的溶液的方法,该方法包括提供冻干形式的多肽(或混合物)并用合适的运载体或稀释剂(例如水)重构。
多肽可以使用本领域已知的方法固定,例如Datta S et al.,Enzymeimmobilization:an overview on techniques and support materials,3Biotech,3(1):1-9(2013)中所述。例如,可以通过吸附、共价结合、亲和力固定或包封来固定多肽。可用作支持物的材料包括但不限于,例如天然支持物(例如琼脂糖(agarose)、胶原、明胶、纤维素、果胶、琼脂糖凝胶)、无机材料(例如陶瓷、二氧化硅、玻璃、活性炭或木炭)或合成聚合物。例如,多肽可以固定在琼脂糖凝胶上。
唾液酸酶
功能特点
除了使用化学和遗传方法修饰聚糖链外,几种酶(糖苷酶)还可以作用于将唾液酸与其他聚糖连接的键。这些被称为唾液酸酶或神经氨酸酶的酶对特定类型的唾液酸键显示出高度的特异性。人糖蛋白中通常存在三种不同的键类型,α(2-3)键为主要形式,其次是α(2-6)和α(2-8)。为了简单起见,这些键类型在本文中可以被称为2-3、2-6和2-8键。2-3键是指唾液酸己糖第2位碳原子通过氧原子与连接的聚糖的己糖第3位碳相连接。相应地,2-6键或2-8键表示分别与连接的聚糖的己糖的第6位或第8位碳相连接于。
大多数已知的唾液酸酶对2-3键具有特异性(以非常高的活性切割该键),或能够切割更宽范围的键,通常切割所有2-3、2-6和2-8键。这些不同类型的唾液酸酶可以分别称为窄谱或广谱。广谱唾液酸酶通常表现出对2-3键的活性高,对2-6键的活性降低,而对2-8键的活性非常低。在该领域中,能有效切割2-8键的酶相对稀少(甚至未知)。
唾液酸酶的酶活性可以通过任何合适的方法来评估,例如实施例中描述的那些。合适的方法可以包括将已知或疑似的唾液酸酶与标准的唾液酸酶底物,例如共同包含2-3、2-6和2-8型键的一个或多个小分子一起孵育。这样的小分子包括2-3'-唾液乳糖、2-6'-唾液乳糖和多聚唾液酸(2-8')。唾液酸酶对这类分子的活性将产生可以通过常规方法对游离唾液酸进行定量。可供选择地,可以使用糖蛋白作为底物来评估唾液酸酶活性。可以使用常规方法(例如SDS-PAGE或凝集素印迹)来检测任何所得的切割产物并对任何所得的切割产物进行定量。
结构特点
本发明人已经从共生肠道菌嗜粘蛋白艾克曼菌中鉴定出并表征了几种唾液酸酶。唾液酸酶之一,在本文中称为Am0707,对2-8键具有出乎意料的高活性,而且也可以切割2-3和2-6键。因此可以认为Am0707是广谱唾液酸酶。另一种唾液酸酶,在本文中称为Am1757,仅对2-3键具有高活性。因此可以认为Am1757是窄谱唾液酸酶。
第一唾液酸酶(Am0707)的完整的野生型一级结构(氨基酸序列)示于SEQ ID NO:1。去除了信号基序的序列示于SEQ ID NO:2。第一唾液酸酶可包括SEQ ID NO:2序列、基本上由SEQ ID NO:2序列组成或由SEQ ID NO:2序列组成,并且通常不超过400个氨基酸。
第二唾液酸酶(Am1757)的完整的野生型一级结构(氨基酸序列)示于SEQ ID NO:4。去除了信号基序的序列示于SEQ ID NO:5。第二唾液酸酶可包括SEQ ID NO:5序列、基本上由SEQ ID NO:5序列组成或由SEQ ID NO:5序列组成,并且通常不超过600个氨基酸。
可供选择地,所述第一唾液酸酶和/或所述第二唾液酸酶可以各自独立地被其各自的变体替代,条件是保留酶活性。所述唾液酸酶的变体可以分别包含以下、基本上由以下组成或由以下组成:SEQ ID NO:2或5序列的氨基酸序列的与所述氨基酸序列至少50%一致的变体。变体序列可以与所述氨基酸序列至少60%、至少70%、至少80%、至少85%、至少90%、至少95%、至少98%或至少99%一致。一致性水平优选为至少85%或更高。相对于序列的一致性可以在序列的至少100、至少200、至少300、至少350、至少400或至少500或更多个连续氨基酸的区域上测量,或更优选在序列的全长上测量。变体的长度通常比参考序列长或短不超过50个氨基酸,并且长度优选与参考序列大约(或完全)相同。
可以使用任何合适的算法来计算氨基酸一致性。例如,PILEUP和BLAST算法可用于计算一致性或将序列对齐(例如识别等效序列或相应序列(通常在其默认设置上)),如在Altschul S.F.(1993)J Mol Evol 36:290-300;Altschul,S,F et al(1990)J Mol Biol215:403-10中所描述。进行BLAST分析的软件可通过美国国家生物技术信息中心(NationalCenter for Biotechnology Information;http://www.ncbi.nlm.nih.gov/)公开获得。该算法涉及首先通过在与数据库序列中相同长度的字比对时在匹配或满足一些正值阈值得分T的查询序列中鉴定长度为W的短字来鉴定高评分序列对(HSP)。T被称为邻近字得分阈值(Altschul et al,同上)。这些最初的邻近字命中充当启动搜索以查找包含它们的HSP的种子。字命中沿着每个序列在两个方向上延伸,直到可以增加累积比对得分为止。在以下情况下,停止在每个方向上的字命中扩展:累积比对得分从其最大实现值下降了数量X;由于一个或多个负得分残基比对的累积,累积得分变为零或更低;或到达任一序列的末尾。BLAST算法参数W、T和X确定比对的灵敏度和速度。BLAST程序使用默认值字长(W)为11,BLOSUM62评分矩阵(请参见Henikoff and Henikoff(1992)Proc.Natl.Acad.Sci.USA 89:10915-10919)比对(B)为50,期望(E)为10,M=5,N=4,以及对两条链的比较。
BLAST算法对两个序列之间的相似性进行统计分析。参见例如Karlin andAltschul(1993)Proc.Natl.Acad.Sci.USA 90:5873-5787。BLAST算法提供的一种相似性度量是最小总和概率(P(N)),最小总和概率(P(N))提供偶然会发生两个多核苷酸或氨基酸序列匹配的概率的指示。例如,如果第一序列与第二序列相比的最小总和概率小于约1,优选小于约0.1,更优选小于约0.01,并且最优选小于约0.001,则认为该序列与另一个序列相似。可供选择地,UWGCG软件包提供BESTFIT程序,该程序可用于计算一致性(例如以其默认设置使用)(Devereux et al(1984)Nucleic Acids Research 12,387-395)。
唾液酸酶的序列可以包含相对于各SEQ ID NO序列进行了修饰(例如氨基酸添加、缺失或取代)的所述SEQ ID NO的变体。除非另有说明,修饰优选是保守氨基酸取代。保守取代用具有相似化学结构、相似化学性质或相似侧链体积的其他氨基酸来替代氨基酸。所引入的氨基酸可以具有与它们所替代的氨基酸相似的极性、亲水性、疏水性、碱性、酸性、中性或电荷。可供选择地,保守取代可以引入另一种芳族或脂族氨基酸替代现有的芳族或脂族氨基酸。保守的氨基酸变化是本领域熟知的,并且可以根据以下表A1中定义的20种主要氨基酸的性质进行选择。在氨基酸具有相似的极性的情况下,可以通过参考表A2中氨基酸侧链的亲水性等级来确定。本发明的唾液酸酶序列可以包含各SEQ ID NO进行了多达10、20、30、40、50或60个保守取代的变体。
表A1-氨基酸的化学性质
表A2-亲水性等级
可供选择地,可以用如上所述的各SEQ ID NO或其变体的较短片段替代唾液酸酶。该片段可以描述为所述SEQ ID NO的保留酶活性的截短形式。这样的片段比相应的SEQ IDNO短,并且长度通常为至少100、150、200、250、300、350、400、450或500个氨基酸。
本文所述的任何唾液酸酶可任选地在N末端包括另外的甲硫氨酸和在C末端包括组氨酸或其他标签。这样的另外的序列可有助于表达和/或纯化。组氨酸标签优选由六个组氨酸残基组成。组氨酸标签优选通过接头连接至C末端,接头通常是短氨基酸序列(例如3至5个氨基酸)。接头通常主要由甘氨酸和丝氨酸残基组成,并且可以优选地包括序列GSG。例如,GSG和GSGLE是合适的接头。
因此,总的来说,第一唾液酸酶是:
(a)包含以下或由以下组成的多肽:SEQ ID NO:2氨基酸序列;
(b)包含以下或由以下组成的多肽:与SEQ ID NO:2氨基酸序列至少85%一致的氨基酸序列;或
(c)包含以下氨基酸序列或由以下氨基酸序列组成的多肽:该氨基酸序列为SEQID NO:2序列的片段或与SEQ ID NO:2氨基酸序列85%一致的氨基酸的片段;
第二唾液酸酶是:
(d)包含以下或由以下组成的多肽:SEQ ID NO:5氨基酸序列;
(e)包含以下或由以下组成的多肽:与SEQ ID NO:5氨基酸序列至少85%一致的氨基酸序列;或
(f)包含以下氨基酸序列或由以下氨基酸序列组成的多肽:该氨基酸序列为SEQID NO:5序列的片段或与SEQ ID NO:5氨基酸序列85%一致的氨基酸的片段;
任选地,其中所述第一唾液酸酶和/或所述第二唾液酸酶在N末端包括另外的甲硫氨酸和/或在C末端包括组氨酸标签,该标签可以通过接头与C末端连接。
示例性的第一唾液酸酶是由SEQ ID NO:3氨基酸序列组成的多肽。示例性的第二唾液酸酶是由SEQ ID NO:6氨基酸序列组成的多肽。
唾液酸酶组合物
唾液酸酶组合物包含至少一种唾液酸酶,优选为基本上分离的或纯化的形式。如上文涉及多肽的一般公开中所述,这通常是指从存在于表达唾液酸酶的细胞的细胞提取物的大部分其他成分中分离。通过基本上纯化,将理解的是,唾液酸酶被纯化至至少50%、60%、70%、80%或优选至少90%同质性。纯度水平可通过任何合适的方法进行评估,但通常涉及样品的SDS-PAGE分析,然后进行考马斯蓝检测。唾液酸酶可以与不会干扰唾液酸酶预期目的的运载体、稀释剂或防腐剂混合,并且仍被为是基本上分离的或纯化的。唾液酸酶组合物可以包括另外的活性成分,例如另一种唾液酸酶或另一种酶,在这种情况下,将每种所述成分分别纯化至高水平的同质性,然后以适当的比例混合以用于各自的预期目的。在本发明优选的唾液酸酶组合物中,所述组合物包含各自纯化至至少90%的同质性并且彼此以1:1的比例存在的第一唾液酸酶和第二唾液酸酶。这样的组合物可以包含另外的活性成分,例如不是唾液酸酶的另一种酶。其他酶可以是蛋白酶和/或糖苷酶。蛋白酶优选是O-糖蛋白特异性内切蛋白酶。糖苷酶优选是O-糖苷酶。两种类型的酶将在下文更详细地讨论。
当唾液酸酶组合物包含不是唾液酸酶的活性成分时,总唾液酸酶含量(例如第一唾液酸酶加上第二唾液酸酶)相对于其它酶的优选比例为1:1。例如,如果组合物包含2000单位的另一种酶,则该组合物还将包含2000单位的唾液酸酶,其中如果存在两种唾液酸酶,则所述2000单位包括1000单位的第一唾液酸酶和1000单位的第二唾液酸酶。
唾液酸酶组合物(通常与多肽一样)可以以适于在使用前在水溶液中重构的冻干形式提供。冻干的组合物具有改善的稳定性,使得能够更长久地储存唾液酸酶。本文提供了一种制备冻干形式的唾液酸酶组合物的方法,所述方法包括在合适的缓冲液(例如Tris-缓冲盐水(TBS))中冷冻干燥一种或多种唾液酸酶。缓冲液优选包含低浓度的NaCl,通常高达300mM、250mM、200mM或150mM。NaCl浓度优选为约150mM,例如在125mM至175mM之间。唾液酸酶通常在冷冻干燥之前是基本上纯化的。还提供了冻干形式的所得组合物。还提供了一种制备唾液酸酶组合物的方法,该唾液酸酶组合物为溶液,该方法包括提供冻干形式的组合物并用合适的运载体或稀释剂(例如水)重构。
本发明人确定第一唾液酸酶对2-8键具有异常高的活性。因此,本发明提供了包含第一唾液酸酶的组合物,所述第一唾液酸酶独立地选自:
(a)包含以下或由以下组成的多肽:SEQ ID NO:2氨基酸序列;
(b)包含以下或由以下组成的多肽:与SEQ ID NO:2氨基酸序列至少85%一致的氨基酸序列;或
(c)包含以下氨基酸序列或由以下氨基酸序列组成的多肽:该氨基酸序列为SEQID NO:2序列的片段或与SEQ ID NO:2氨基酸序列85%一致的氨基酸的片段;
任选地,其中所述第一唾液酸酶在N末端包括另外的甲硫氨酸和/或在C末端包括组氨酸标签,该标签可以通过接头与C末端连接。
所述组合物可以用于优选以高效率切割2-8唾液酸键的方法中。这样的组合物的实例包含由SEQ ID NO:3氨基酸序列组成的唾液酸酶。
本发明人还确定了第一唾液酸酶(Am0707)和第二唾液酸酶(Am1757)的组合以异常高的效率水解2-3、2-6和2-8键,从而允许有效去除任何糖蛋白的基本上所有(通常大于90%)的唾液酸。该组合还对天然(非变性)状态的糖蛋白具有令人惊讶的效果。因此,本发明提供如上所述的包含第一唾液酸酶的组合物,该组合物还包含第二唾液酸酶,第二唾液酸酶独立地选自:
(d)包含以下或由以下组成的多肽:SEQ ID NO:5氨基酸序列;
(e)包含以下或由以下组成的多肽:与SEQ ID NO:5氨基酸序列至少85%一致的氨基酸序列;或
(f)包含以下氨基酸序列或由以下氨基酸序列组成的多肽:该氨基酸序列为SEQID NO:5序列的片段或与SEQ ID NO:5氨基酸序列85%一致的氨基酸的片段。
任选地,其中所述第二唾液酸酶在N末端包括另外的甲硫氨酸和/或在C末端包括组氨酸标签,该标签可以通过接头与C末端连接。第一唾液酸酶和第二唾液酸酶优选相对于彼此以1:1的比例存在。
所述组合物可以用于优选以高效率使糖蛋白完全去唾液酸化或切割糖蛋白中大于90%的唾液酸键的方法中。糖蛋白优选为天然状态。也就是说,它没有经历任何形式的变性条件。
本发明的唾液酸酶组合物的实例包含由SEQ ID NO:3氨基酸序列组成的唾液酸酶和由SEQ ID NO:6氨基酸序列组成的唾液酸酶,优选比例为1:1。
唾液酸酶组合物的唾液酸酶活性可以使用与上文针对单独的唾液酸酶所述相同的方法评估。但是,优选使用非变性糖蛋白作为底物进行评估。当底物与示例性唾液酸酶或其混合物(例如由SEQ ID NO:3氨基酸序列组成的多肽和由SEQ ID NO:6氨基酸序列组成的多肽的1:1混合物)接触时,可以将结果与在相同测定中获得的结果进行比较。一个单位的这样的唾液酸酶混合物通常是如SDS-PAGE所监测的在20mM Tris pH 6.8中于37℃下孵育2小时时,从大于等于90%的1μg糖蛋白(胎球蛋白)中水解唾液酸所需的量。这被认为代表高效率。
O-糖苷酶
本发明人也已经鉴定出并表征了来自存在于哺乳动物口道中的共生细菌口腔链球菌的O-糖苷酶,该酶特别是在与如上所述的唾液酸酶组合物组合使用时有效地水解O-连接的聚糖。O-糖苷酶在本文中可以被称为“O-glyk”或“So”。野生型O-glyk序列以SEQ IDNO:7提供,该序列包括信号序列和LPTXG细胞壁锚定基序。缺少信号序列的野生型O-glyk序列以SEQ ID NO:8提供。缺少信号序列和细胞壁锚定基序的C末端部分的野生型O-glyk序列以SEQ ID NO:9提供。可以任选地修饰这些序列,以在N末端包括另外的甲硫氨酸和/或在C末端包括组氨酸或其他标签。这样的另外的序列可以帮助表达(例如在大肠杆菌中)和/或纯化。组氨酸标签优选由六个组氨酸残基组成。组氨酸标签优选通过接头连接至C末端,接头通常是短氨基酸序列(例如3至5个氨基酸)。接头通常主要由甘氨酸和丝氨酸残基组成,并且可以优选地包括序列GSG。例如,GSG和GSGLE是合适的接头。在N末端具有另外的甲硫氨酸且在C末端具有GSGLE接头和His6标签的示例性O-glyk序列以SEQ ID NO:10提供。本发明中任何对“O-glyk”或“So”的提及都可以表示SEQ ID NO:7、8、9或10中的任一种,但优选地是指通长不长于2070个氨基酸并且包含SEQ ID NO:9氨基酸序列或由SEQ ID NO:9氨基酸序列组成的多肽。最优选的是由SEQ ID NO:10氨基酸序列组成的多肽。
本发明人还发现如上所述的唾液酸酶组合物的作用还增强了其他O-糖苷酶的活性。因此,本发明还提供了修饰糖蛋白的方法,该方法包括使糖蛋白样品与上述唾液酸酶组合物和O-糖苷酶两者接触。本发明还提供了如上所述的唾液酸酶组合物,该唾液酸酶组合物任选地还包含O-糖苷酶。在所述方法和所述组合物中,所述O-糖苷酶可以包含SEQ IDNO:9氨基酸序列或由SEQ ID NO:9氨基酸序列组成,或者可以是任何其他O-糖苷酶,例如从肠细菌(例如粪肠球菌(Enterococcus faecalis))获得的酶。来自粪肠球菌的优选的O-糖苷酶是具有Uniprot条目B5UB72版本22的氨基酸序列的多肽。其他合适的O-糖苷酶包括在WO2009129086中描述的那些,特别是如WO2009129086第7页和图5所示的EngEF、EngPA和截短的EngAA。
在本文的任何公开中,包含SEQ ID NO:9序列的O-糖苷酶可以被其变体替代,条件是保留酶活性。O-糖苷酶的变体可以包含SEQ ID NO:9序列的氨基酸序列的变体,基本上由SEQ ID NO:9序列的氨基酸序列的变体组成或由SEQ ID NO:9序列的氨基酸序列的变体组成。除了要保留的相关酶活性是针对O-聚糖的水解活性之外,所述SEQ ID NO的变体可以如上文关于唾液酸酶的陈述所定义。
可供选择地,O-糖苷酶可被如上所述的SEQ ID NO:9或其变体的较短片段替代。该片段可以描述为所述SEQ ID NO的保留酶活性的截短形式。这样的片段比SEQ ID NO:3短,并且长度通常为至少300、400、500、600、800、1000、1200、1300、1400或1500个氨基酸。
本文所述的任何O-糖苷酶可任选地在N末端包括另外的甲硫氨酸和/或在C末端包括组氨酸或其他标签。这样的另外的序列可有助于表达和/或纯化。组氨酸标签优选由六个组氨酸残基组成。组氨酸标签优选通过接头连接至C末端,所述接头通常是短氨基酸序列(例如3至5个氨基酸)。接头通常主要由甘氨酸和丝氨酸残基组成,并且可以优选地包括序列GSG。例如,GSG和GSGLE是合适的接头。这种类型的示例性O-糖苷酶由SEQ ID NO:10氨基酸序列组成。
O-糖苷酶的酶活性可以通过任何合适的方法来评估,例如实施例中描述的那些。合适的方法可以包括将已知或疑似的O-糖苷酶与标准底物(例如一个或多个共同包含O-聚糖核心区域的小分子)一起孵育。这样的小分子包括4-甲基伞形酮(4MU)底物和pNP底物,pNP的释放表明其活性。可供选择地,可以使用糖蛋白作为底物评估活性。可以使用常规方法(例如SDS-PAGE或凝集素印迹)检测任何所得的切割产物并对任何所得的切割产物进行定量。在将糖蛋白用作底物的情况下,可能需要用如上所述的唾液酸酶组合物进行预处理(或同时处理)。将底物与示例性O-糖苷酶(例如由SEQ ID NO:10氨基酸序列组成的多肽)接触时,可以将结果与在相同试验中获得的结果进行比较。一个单位的SEQ ID NO:10的多肽被定义为如SDS-PAGE所监测的在20mM Tris pH 6.8中于37℃下孵育2小时时,与一个单位的唾液酸酶混合物(优先的唾液酸酶混合物如上所述)组合从大于等于90%的1μg糖蛋白(胎球蛋白)中去除O-聚糖所需的量。测试多肽优选在以相同的量存在时达到相似的活性水平。在实施例中也描述了示例性测定。
可以以溶液或冻干形式提供包含如上所述的O-糖苷酶的组合物以在溶液中重构。可将O-糖苷酶冻干在pH 7.6的Tris缓冲盐水中。
O-聚糖特异性内切蛋白酶
本发明人还发现如上所述的唾液酸酶组合物的作用增强了O-聚糖特异性内切蛋白酶的活性,特别是包含SEQ ID NO:11氨基酸序列的O-聚糖特异性内切蛋白酶的活性,该O-聚糖特异性内切蛋白酶通常不长于375个氨基酸,并且优选由SEQ ID NO:12氨基酸序列组成的多肽。
因此,本发明还提供了修饰糖蛋白的方法,该方法包括使糖蛋白样品与如上所述的唾液酸酶组合物以及与O-聚糖特异性内切蛋白酶二者接触。本发明还提供了如上所述的唾液酸酶组合物,该组合物任选地还包含O-聚糖特异性内切蛋白酶。在所述方法和所述组合物中,所述O-聚糖特异性内切蛋白酶可以是SEQ ID NO:12的O-聚糖特异性内切蛋白酶。
在本文的任何公开内容中,倘若保留酶活性,SEQ ID NO:11的O-聚糖特异性内切蛋白酶可以被其变体替代。内切蛋白酶的变体可以包含SEQ ID NO:11序列的氨基酸序列的变体,基本上由SEQ ID NO:11序列的氨基酸序列的变体组成或由SEQ ID NO:11序列的氨基酸序列的变体组成。除了要保留的相关酶活性是针对O-聚糖的水解活性之外,所述SEQ IDNO的变体可以如上文关于唾液酸酶的陈述所定义。
可供选择地,O-糖苷酶可被如上所述的SEQ ID NO:3或其变体的较短片段替代。该片段可以描述为所述SEQ ID NO的保留酶活性的截短形式。这样的片段比SEQ ID NO:11短,并且长度通常为至少100、150、200、250、300、350、400、450或500个氨基酸。
本文所述的任何内切蛋白酶可任选地在N末端包括另外的甲硫氨酸和/或在C末端包括组氨酸或其他标签。这样的另外的序列可有助于表达和/或纯化。组氨酸标签优选由六个组氨酸残基组成。组氨酸标签优选通过接头连接至C末端,该接头通常是短氨基酸序列(例如3至5个氨基酸)。接头通常主要由甘氨酸和丝氨酸残基组成,并且可以优选地包括序列GSG。例如,GSG和GSGLE是合适的接头。这种类型的示例性O-糖苷酶由SEQ ID NO:12氨基酸序列组成。
内切蛋白酶的酶活性可以通过任何合适的测定来评估。例如,可以将标准的O-糖蛋白底物(例如IgA分子)与测试多肽一起孵育。然后可以通过SDS-PAGE和/或质谱来分析起始材料和反应产物,以确定切割产物(如果有的话)的存在,并且如果需要还可以进一步表征那些产物。未O-糖基化的糖蛋白底物(例如IgG1分子)可以用作阴性对照。当底物与示例性多肽(例如由SEQ ID NO:12氨基酸序列组成的多肽)接触时,可以将结果与在相同测定中获得的结果进行比较。
可以以溶液或冻干形式提供包含如上所述的O-聚糖特异性内切蛋白酶的组合物以在溶液中重构。可将O-聚糖特异性内切蛋白酶冻干在pH 7.6的Tris缓冲盐水中。
使用方法
本发明提供了将样品与本发明的组合物在适合于所述组合物中的酶作用于所存在的任何底物的条件下孵育的任何方法。所述方法可以任选地包括对所得产物的分析。所述分析可包括通过任何合适的手段来分开和/或检测和/或分离产物,该合适的手段包括SDS-PAGE、HPLC、凝集素印迹、ELISA或质谱。
合适的条件包括与本发明的组合物孵育至少20分钟、30分钟、40分钟、50分钟、60分钟、70分钟、80分钟、90分钟或120分钟、3小时、5小时、10小时、12小时或过夜。孵育优选在室温下进行,更优选在约20℃、25℃、30℃、35℃、40℃或45℃下,最优选在约37℃下进行。该方法可以在任何合适的pH下进行。合适的pH值包括例如约3.0、3.5、4、4.5、5、5.5、6、6.5、7、7.5、8、8.5、9或9.5的pH。优选的pH在5.6至6.8的范围内。该方法可以在任何合适的缓冲液中进行,例如Tris缓冲盐水(TBS)或磷酸盐缓冲盐水(PBS)。缓冲液优选包含低浓度的NaCl,通常不超过300mM、250mM、200mM或150mM。NaCl浓度优选为约150mM,例如在125至175mM之间。
本发明的组合物中的酶与样品的蛋白质含量的近似比例可以是1:1、2:1、4:1、6:1、10:1、15:1、20:1、1:2、1:4、或1:6、1:10、1:15、1:20、1:40、1:50或1:100。
以下是本发明特别优选的方法:
一种用于修饰糖蛋白的方法,该方法包括使含有糖蛋白的样品与本发明的组合物接触,并任选地分析所得产物。所述分析可以包括通过任何合适的方法来分开和/或检测和/或分离产物,该合适的方法包括SDS-PAGE、HPLC、凝集素印迹、ELISA或质谱。
在特别的实施方式中,该方法可以包括使样品与仅包含唾液酸酶的本发明的组合物接触,任选地分开产物,然后使所述产物与另一种酶(例如蛋白酶和/或糖苷酶)接触。该方法可描述为在添加其他酶之前的对样品进行去除唾液酸的“预处理”。在该实施方式的变形中,其他酶可以与唾液酸酶组合物分开地但同时地添加到样品中,并且可以被描述为“共处理”。在另一种变形中,其它酶存在于唾液酸酶组合物中。其它酶优选是例如本文所述的O-聚糖特异性内切蛋白酶或O-糖苷酶。
以下实施例说明了本发明:
实施例1-唾液酸酶
材料与方法
唾液酸酶的表达和纯化
嗜粘蛋白艾克曼菌中鉴定的基因(Am0705、Am0707、Am1757、Am2058)经过密码子优化,从而在载体pET21a(+)中在大肠杆菌中表达良好。该载体被转化到BL21(DE3)Star细胞中。在LB中以37℃、200rpm常规培养大肠杆菌。在质粒存在下,添加100μg/mL卡那霉素。过夜孵育后,将培养物在新鲜的LB(amp)中以1:20稀释,并使培养物生长直至OD620约为0.7至0.8,之后通过添加1mM IPTG诱导重组蛋白表达,并且表达持续5小时,然后收集细胞并冷冻。将冷冻的细胞解冻并溶解在His结合缓冲液(20mM NaP pH 7.4、500mM NaCl,20mM咪唑)中,进行超声处理以释放细胞内蛋白质。通过离心去除细胞碎片。经过滤的无菌上清液在镍柱上进行亲和纯化,并在PD-25柱上重新缓冲至20mM Tris-HCl pH 8.0中。使用Nanodrop确定蛋白质的浓度,并通过SDS-PAGE估算纯度。
使用小分子进行活性评估
使用2-3'-唾液乳糖,2-6'-唾液乳糖和多聚唾液酸(2-8';Sigma-Aldrich)作为底物,以确定酶的水解特异性。将酶(0.05μg)与底物(25μM)在20mM Tris-HCl pH 6.8中混合,并在37℃下孵育30分钟,然后根据制造商的说明对混合物中的游离唾液酸进行定量(唾液酸定量试剂盒,Abcam)。
使用蛋白质底物进行活性评估
将TNFαR、EPO、Enbrel和胎球蛋白(0.5μg)与不同浓度的测试唾液酸酶(1:40)或商业唾液酸酶(来自NEB,根据制造商)一起混合并孵育30分钟,然后将蛋白在4%至20%Novex梯度SDS-PAGE上分离和/或用SNA凝集素印迹进行分析。
凝集素印迹
使用Trans-Blot Turbo转移系统(BioRad)将分开的蛋白质转移到PVDF膜上。用凝集素缓冲液封闭膜,然后将膜与主要结合剂(SNA-生物素)和次要结合剂(HRP-链霉亲和素;VectorLabs)一起孵育,并且在其间进行洗涤步骤。通过West Pico SuperSignal(ThermoFisher)试剂盒使化学发光试剂显色,并在ChemiDoc(BioRad)中进行了检测。
最佳酶促条件
将唾液酸酶与20mM Tris-HCl pH 8.0中的2-3'唾液酸乳糖(25μM)孵育,以研究NaCl(0至1.5M)和离子(2mM CaCl2、2mM ZnCl2、5mM EDTA)的影响。为了最佳的pH,将唾液酸酶在乙酸缓冲液(4.6和5.6)和Tris-HCl缓冲液(6.8、7.4、8.0和8.8)中孵育。根据制造商的说明,将所有样品在室温下孵育15分钟,然后将混合物以1:1添加到唾液酸定量试剂盒(Abcam)中。所有值均表示为相对于每个组中最高活性的相对活性。
与知名生物技术公司的已知唾液酸酶比较
根据制造商的说明,将嗜粘蛋白艾克曼菌唾液酸酶、唾液酸酶混合物和购自知名品牌的唾液酸酶(NEB P0743S、P0720S和P0722S)与2-3'和2-6'唾液乳糖和多聚唾液酸在其各自的最佳缓冲液连同唾液酸定量缓冲液(1:1比例)中孵育20分钟。
结果
所有嗜粘蛋白艾克曼菌唾液酸酶均表达良好并且可在His柱上纯化
艾克曼菌标注的唾液酸酶Am0705、Am0707、Am1758和Am2058在大肠杆菌中重组表达,并在His柱上纯化至高纯度(图1)。蛋白质表达良好,每L大肠杆菌培养物中的纯化酶水平在70至150mg之间变化。此外,唾液酸酶在Tris缓冲液中高度溶解,并且可以浓缩至大于3mg/mL而无明显沉淀。
唾液酸酶中仅三种显示出对多种蛋白质底物的活性
在针对不同的O-连接糖蛋白和N-连接糖蛋白对纯化唾液酸酶进行初步筛选期间,除Am0705以外的所有唾液酸酶均显示有效活性(数据未显示)。由于Am2058的活性不一致,我们继续表征Am0707和Am0707/1757(混合物,1:1)的混合物。
唾液酸酶具有不同的特异性
几种唾液酸酶显示出键特异性,并且水解某些唾液酸键(例如2-3、2-6和/或2-8)的能力有限。为了研究四种嗜粘蛋白艾克曼菌唾液酸酶作用于不同键的能力,我们将唾液酸酶与仅呈现一种键类型的特定底物一起孵育(2-3'-唾液乳糖、2-6'-唾液乳糖和多聚唾液酸),并对游离唾液酸进行定量(图2)。Am1757对2-3键具有较高的比活性,而Am0707对所有测试的键具有较宽(但较低)的活性。Am0707和Am1757的组合(混合物)产生了出色的广谱产品(图2)。
唾液酸酶对NaCl敏感并且依赖于二价阳离子
为了进一步研究优化唾液酸酶活性所需的条件,我们研究了唾液酸酶对离子、pH和NaCl的依赖性。两种唾液酸酶的表现相似,对EDTA和Zn2+的敏感度高,同时依赖于Ca2+。该酶在中性至碱性pH值下具有较高的活性,并且在NaCl存在下丧失了大部分活性(图3)。
唾液酸酶混合物提高整体水解效率
由于表征的唾液酸酶具有互补活性,其中Am1757具有2-3键高水解活性,而Am0707还对有2-6、2-8键起作用,因此我们通过改变两种酶的比例研究了两种酶的混合物是否可以显示出对天然糖蛋白上的所有唾液酸键的高效率。包含1:40Am1757和1:40Am0707的混合物迅速(<15分钟)水解胎球蛋白上的所有唾液酸键(图4)。
唾液酸酶针对小分子底物的基准测试
为了比较唾液酸酶混合物(GVS_Smix)与单一酶以及与品牌竞争对手的效率,我们将我们的酶与来自New England Biolabs(NEB)的三种可商购唾液酸酶进行了比较。这些是来自产脲节杆菌(Arthrobacter ureafaciens)的广谱唾液酸酶(NEB A,来自产脲节杆菌的酶α2-3、α2-6、α2-8、α2-9神经氨酸酶A,目录号P0722S;切割2-3、2-6、2-8和2-9键)、来自肺炎链球菌(Streptococcus pneumoniae)的窄谱唾液酸酶(NEB S,来自肺炎链球菌的α2-3神经氨酸酶S,目录号P0743S;仅切割2-3键)和来自产气荚膜梭菌(Clostridiumperfringens)的一般唾液酸酶(NEB O,来自产气荚膜梭菌的酶α2-3、α2-6、α2-8神经氨酸酶O,目录号P0720S,切割2-3、2-6、2-8键)。按照制造商的建议添加酶,并与底物在37℃下孵育30分钟(图5)。
通过SDS-PAGE判断出,GVS_Smix中不同样品中的唾液酸酶的量低于NEB中的量,而神经氨酸酶A可能是例外。神经氨酸酶S展示出对糖蛋白上的所有唾液酸键的水解能力有限,而神经氨酸酶A和神经氨酸酶O水解存在于胎球蛋白上的所有唾液酸(图6)。同样,正如对针对小分子合成底物的活性的观察,Smix水解糖蛋白上的所有唾液酸键,产生窄的蛋白带,表明了完全去唾液酸化。Am1757处理过的样品无法水解2-6或2-8键,相比用Smix处理过的那些样品,作为更高的分子量迁移。通过SNA印迹、标记唾液酸证实了水解,表明所有广谱唾液酸酶均有效去除了存在于胎球蛋白上的所有唾液酸,而2-3特异性的酶(例如神经氨酸酶S和Am1757)仅去除了一部分唾液酸(图6)。
Smix可以从天然蛋白质中有效释放唾液酸
即使Smix可以以与NEB产品相似或更好的效率作用于小的半合成底物(图5),也必须研究Smix对具有不同唾液酸键的天然蛋白的活性。选择胎球蛋白作为模型蛋白,因为胎球蛋白同时具有2-3和2-6键合的唾液酸。使用SNA印迹,很明显的是,与NEB A和NEB O相似,所选浓度的Smix可以从胎球蛋白中完全去除所有唾液酸(图6)。正如所预期的,两种2-3特异性酶Am1757和NEB S无法完全将胎球蛋白去唾液酸化。
为了更定量地确定Smix和Am1757与三种NEB唾液酸酶相比从天然蛋白质释放唾液酸的能力,我们将不同的糖蛋白底物(TNFaR、IgA、纤溶酶原和阿巴西普[Orencia])与每种唾液酸酶孵育15+15分钟并定量释放的唾液酸。尽管某些底物被证实比其他底物更难水解,但在所有情况下,GVS_Smix至少与NEB产品相当(图7),并显示出最高的一致性,因为GVS_Smix对所有底物均表现出高活性。尽管单独的Am1757酶和NEB产品中的每一种针对一种或另一种底物均具有较高的活性,但只有GVS_Smix对所有底物均一致地显示出高活性,使GVS_Smix成为用于聚糖分析的极具吸引力的工具。
实施例2-O-糖苷酶
材料和方法
内-O-糖苷酶的表达和纯化
口腔链球菌的内-N-乙酰基半乳糖胺酶经过密码子优化,从而在载体pET21a(+)中在大肠杆菌中表达良好。该载体被转化到BL21(DE3)Star细胞中。在LB中以37℃、200rpm常规培养大肠杆菌。在质粒存在下,添加100μg/mL卡那霉素。过夜孵育后,将培养物在新鲜的LB(amp)中以1:20稀释,并使培养物生长直至OD620约为0.7至0.8,之后通过添加1mM IPTG诱导重组蛋白表达,并且表达持续5小时,然后收集细胞并冷冻。将冷冻的细胞解冻并溶解在His结合缓冲液(20mM NaP pH 7.4、500mM NaCl,20mM咪唑)中,进行超声处理以释放细胞内蛋白质。通过离心去除细胞碎片。经过滤的无菌上清液在镍柱上进行亲和纯化,并在PD-25柱上重新缓冲至PBS中。使用Nanodrop测定蛋白质的浓度,并通过SDS-PAGE估算纯度。蛋白质的序列以SEQ ID NO:3提供。
使用小分子进行活性评估
使用O-聚糖核心区域的4-甲基伞形酮(4MU)底物和pNP底物作为底物,以确定酶的水解活性。将酶(1μg)与底物(2mM)混合,并在37℃下孵育15至120分钟,在此期间分别记录荧光和吸光度(405nm)。
使用蛋白质底物进行活性评估
在存在或不存在Smix(1:40+1:40)或Am1757(1:40)的情况下,将TNFαR、EPO、Enbrel、胎球蛋白、IgA、Orencia和纤溶酶原(0.5μg)与O-糖苷酶混合(1:40)0至24小时。将蛋白在4至20%Novex梯度SDS-PAGE上分开。
最佳酶促条件
将酶与其各自的底物(4MU或pNP)孵育,以研究NaCl(0至1.5M)和离子(2mM CaCl2、2mM ZnCl2、5mM EDTA)依赖性。为了最佳的pH,将酶在乙酸缓冲液(50mM pH 4.6和5.6)和Tris-HCl缓冲液(20mM pH 6.8、7.4、8.0和8.8)中孵育。将所有样品在37℃下孵育15分钟。所有值均表示为相对于每个组中最高活性的相对活性。
与可商购的酶的比较
将鉴定出的酶以及来自知名品牌的可商购的酶(来自粪肠球菌的NEB O-糖苷酶,商品目录号P0733S,也作为捆绑商品#E0540S)与它们各自的底物和优化的缓冲液一起孵育,并在天然条件下与不同的糖蛋白孵育0-24小时,然后在SDS-PAGE上分开。
结果
口腔链球菌内-α-N-乙酰基半乳糖胺酶的表达/纯化
在发现阶段,考虑了来自口腔链球菌和两歧双歧杆菌的两种不同的内-α-N-乙酰半乳糖胺酶,两者均为高分子量细菌蛋白(>200kDa)。两歧双歧杆菌糖苷酶高度不稳定,或至少在表达和亲和纯化后导致高度片段化的部分。尽管对于口腔链球菌也可以看到某些片段化,但是这种片段化是很不明显的(图8A)。另外,该酶在4℃下稳定达一周而没有任何其他降解(图8B)。但是,表达水平(约5至10mg/L)非常低。进一步的分析表明,片段化归因于超声处理。
O-糖苷酶可以作用于合成的pNP标记的核心1至3O-聚糖。
对这两种O-糖苷酶的继续分析显示了对核心1聚糖的显著偏好,而对核心2和3的活性要低得多(图9)。重要的是,来自口腔链球菌的糖苷酶显示出比来自两歧双歧杆菌的相应基因显著更高的活性,因此我们决定主要关注口腔链球菌O-糖苷酶。
添加MgCl2显著增加口腔链球菌O-糖苷酶的活性
为了确定O-糖苷酶的最佳条件,将酶与pNP核心1底物在不同的条件下,在一定范围的pH和离子下孵育(图10)。在中性至弱碱性pH值下显示高活性的O-糖苷酶被Zn2+完全抑制,但在高达8mM MgCl2的存在下具有增强的活性(图10C)。
口腔链球菌的O-糖苷酶可以水解天然糖蛋白中的聚糖
为了研究介导所有O-聚糖水解所需的动力学和剂量,将天然TNFαR与不同量的O-糖苷酶和实施例1中表征的唾液酸酶混合物(Am1757:Am0707的1:1混合物)的组合孵育1至12小时,或仅与唾液酸酶混合物孵育1至12小时。如图11所示,即使浓度相当低的酶(例如约0.1μg,1:10)也可以在1小时内水解底物。将孵育时间增加到12小时导致该酶与高浓度的唾液酸酶以1:50的比例完全水解底物的能力。重要的是要注意,在某些方面,该酶是一种“无或全部”的酶,几乎不产生半水解的糖蛋白,而是产生未水解或完全水解的蛋白。
口腔链球菌O-糖苷酶可作用于不同的天然糖蛋白
为了进一步研究O-糖苷酶是仅作用于TNFαR还是可以作用于若干种天然糖蛋白,我们将7种不同的糖蛋白与水解酶组合进行了孵育(图12)。口腔链球菌O-糖苷酶,以及两歧双歧杆菌酶(与唾液酸酶组合)能够在孵育过夜后水解所有天然蛋白质,即使对纤溶酶原的活性由于该蛋白的分子量高而很难评估。然而,由于末端唾液酸抑制了活性,所以两种酶确实强烈依赖于唾液酸酶的存在。
与NEB产品系列相比,口腔链球菌O-糖苷酶在水解天然糖蛋白方面是优越的。
为了比较口腔链球菌糖苷酶相比现有商品的活性,我们使用不同量的糖苷酶比较了它们在1小时或16小时内水解TNFαR的能力。使用约1:5的酶:底物比例,口腔链球菌糖苷酶能够在1小时内完全水解其底物。即使在酶与底物的比例为1:1的情况下,NEB O-糖苷酶也不会水解所有O-连接的聚糖,而只会作用于一些容易接近的聚糖。进一步孵育(例如16小时)使酶:底物比例为1:50,同时仍保持口腔链球菌糖苷酶的全部效果。然而,NEB O-糖苷酶仍不能完全使产物去糖基化,表明变性对该酶的功能至关重要,而口腔链球菌O-糖苷酶产物对天然蛋白也具有高活性(图13)。口腔链球菌的O-糖苷酶不仅可以作用于天然蛋白质,而且还能够水解变性蛋白质中的聚糖(数据未显示)。
聚糖组成影响O-糖苷酶的活性
为了进一步研究特定聚糖对O-糖苷酶活性的必要性,在添加O-糖苷酶之前,将TNFαR与不同的酶预孵育以去除单个聚糖。去除末端唾液酸与半乳糖的存在一样对于活性至关重要,这表明O-糖苷酶不能去除单个(末端)GalNAcs(图14)。
O-糖苷酶可高效去除天然蛋白质中的O-聚糖
一旦确定了组合的O-glyk+Smix组合物的最终浓度(1:40),我们便重复与竞争对手品牌产品(NEB)进行比较。虽然使用NEB产品可以检测到对TNFαR的一些水解,但很明显,甚至在12小时后,水解仍未完成。与此相反,在凝集素印迹的支持下,组合的O-glyk+Smix组合物导致糖蛋白在4小时内完全水解(图15)。
同样,Enbrel表现出相同的模式,组合的O-glyk+Smix组合物能够将Enbrel完全水解,而NEB产品则不能。但是,对于胎球蛋白,这两种产品似乎都同样有效(图16)。
2-3连接和2-6连接的唾液酸均抑制O-糖苷酶的活性
为了评估2-3或2-6连接的唾液酸对O-糖苷酶活性的相互影响,我们将O-糖苷酶与2-3特异性唾液酸酶或广谱唾液酸酶(例如分别为Am1757和Smix)一起孵育。尽管初步数据表明Am1757和Am0707可以从糖蛋白释放等量的唾液酸,但是用Am1757(或Smix)处理糖蛋白会导致底物(TNFaR)更快地水解。然而,对于完全水解,至关重要的是用广谱唾液酸酶处理糖蛋白,以同时去除2-6(或2-8)唾液酸(图17)。
GVS O-糖苷酶束的天然活性归因于高效唾液酸酶活性
为了确定各个成分在组合的O-glyk+Smix组合物中的影响,将O-glyk和NEB与四种不同的唾液酸酶产物(GVS_Smix、NEB A、NEB S和NEB O)孵育。组合的O-glyk+Smix组合物有效地水解了来自Enbrel的所有O-聚糖(图18),同时将唾液酸酶换成任何NEB产品会导致O-glyk的活性显著降低,而NEB唾液酸酶A是最有效的。虽然NEB O-糖苷酶束(NEB O-糖苷酶与NEB唾液酸酶O)不能水解Enbrel或TNFαR(图15、16),但是通过使用GVS_Smix代替NEB唾液酸酶O,观察到糖蛋白完全水解,表明组合的O-glyk+Smix组合物的效率至少部分依赖于唾液酸酶水解糖蛋白上所有2-3和2-6键的能力。
实施例3–O-糖蛋白特异性内切蛋白酶
我们最近确定了由SEQ ID NO:12组成的多肽的内切蛋白酶活性至少部分地依赖于特定的唾液酸键,为了全部效果,需要去除2-3和2-6连接的唾液酸。为了确定特定唾液酸键对内切蛋白酶活性的单独的作用,我们将依那西普与不同的唾液酸酶和内切蛋白酶的组合孵育30分钟至20小时。去除2-3键似乎足以增强内切蛋白酶活性(图19)。使用的唾液酸酶是(1)Am0707=SEQ ID NO:3的多肽;(2)Am1757=SEQ ID NO:6的多肽;Mix=(1)和(2)的1:1组合。
序列
SEQ ID NO:1-唾液酸酶,Am0707-野生型(信号序列加有下划线)
MTWLLCGRGKWNKVKRMMNSVFKCLMSAVCAVALPAFGQEEKTGFPTDRAVTVFSAGEGNPYASIRIPALLSIGKGQLLAFAEGRYKNTDQGENDIIMSVSKNGGKTWSRPRAIAKAHGATFNNPCPVYDAKTRTVTVVFQRYPAGVKERQPNIPDGWDDEKCIRNFMIQSRNGGSSWTKPQEITKTTKRPSGVDIMASGPNAGTQLKSGAHKGRLVIPMNEGPFGKWVISCIYSDDGGKSWKLGQPTANMKGMVNETSIAETDNGGVVMVARHWGAGNCRRIAWSQDGGETWGQVEDAPELFCDSTQNSLMTYSLSDQPAYGGKSRILFSGPSAGRRIKGQVAMSYDNGKTWPVKKLLGEGGFAYSSLAMVEPGIVGVLYEENQEHIKKLKFVPITMEWLTDGEDTGLAPGKKAPVLK
SEQ ID NO:2-唾液酸酶,Am0707-无信号的野生型
QEEKTGFPTDRAVTVFSAGEGNPYASIRIPALLSIGKGQLLAFAEGRYKNTDQGENDIIMSVSKNGGKTWSRPRAIAKAHGATFNNPCPVYDAKTRTVTVVFQRYPAGVKERQPNIPDGWDDEKCIRNFMIQSRNGGSSWTKPQEITKTTKRPSGVDIMASGPNAGTQLKSGAHKGRLVIPMNEGPFGKWVISCIYSDDGGKSWKLGQPTANMKGMVNETSIAETDNGGVVMVARHWGAGNCRRIAWSQDGGETWGQVEDAPELFCDSTQNSLMTYSLSDQPAYGGKSRILFSGPSAGRRIKGQVAMSYDNGKTWPVKKLLGEGGFAYSSLAMVEPGIVGVLYEENQEHIKKLKFVPITMEWLTDGEDTGLAPGKKAPVLK
SEQ ID NO:3-唾液酸酶,Am0707-具有另外的N末端甲硫氨酸和C末端接头+His6标签(加粗并加有下划线)
QEEKTGFPTDRAVTVFSAGEGNPYASIRIPALLSIGKGQLLAFAEGRYKNTDQGENDIIMSVSKNGGKTWSRPRAIAKAHGATFNNPCPVYDAKTRTVTVVFQRYPAGVKERQPNIPDGWDDEKCIRNFMIQSRNGGSSWTKPQEITKTTKRPSGVDIMASGPNAGTQLKSGAHKGRLVIPMNEGPFGKWVISCIYSDDGGKSWKLGQPTANMKGMVNETSIAETDNGGVVMVARHWGAGNCRRIAWSQDGGETWGQVEDAPELFCDSTQNSLMTYSLSDQPAYGGKSRILFSGPSAGRRIKGQVAMSYDNGKTWPVKKLLGEGGFAYSSLAMVEPGIVGVLYEENQEHIKKLKFVPITMEWLTDGEDTGLAPGKKAPVLK/>
SEQ ID NO:4-唾液酸酶,Am1757-野生型(信号序列加有下划线)
MKNLLFALLTGSFCCCYAQQKAAPVPEPEVVATPPADAGRGLIRVDSREIRHYSGTRKEPDYLVSRDNGKTWEMKAAPAGYPPNYGGIPKESPAIVRNPLTREFIRVQPIGGFVFLSRGGLDGKWLAVTNDGKLEEDWKDPEKRKNLKKLGGIMRTPVFVNKGRRVIVPFHNMGGGTKFHISDDGGLTWHVSRNGVTSPRHEARPPHQGVRWFNNAVEATVLEMKDGTLWALARTSQDQAWQAFSKDYGETWSKPEPSRFFGTLTMNTLGRLDDGTIVSLWTNTMALPENATAGNGTWEDVFTNRDSHHIAMSGDEGKTWYGFREIILDEHRNHPGYATLDGPEDRGKHQSEMVQLDKNRILISLGQHKNHRRLVIVDRRWVGAKTRATQTGKDLDSQWTIHTYIPQKKGHCSYNRKPSAELVQDPSGGTKKVLQIKRLDDPELVNEKSNVDYRNGGATWNFPNGTTGLVKFRFRVVDGEQADDSGLQVSLTDRLFNACDSTTKDYALFTFPIRLKPAPHLLLGMKKVPFTPGAWHEISLLWQGGQAVVSLDGKKAGTLKMANKSPNGASYIHFISTGSQPDAGILLDTVNARVK
SEQ ID NO:5-唾液酸酶,Am1757-无信号的野生型
QQKAAPVPEPEVVATPPADAGRGLIRVDSREIRHYSGTRKEPDYLVSRDNGKTWEMKAAPAGYPPNYGGIPKESPAIVRNPLTREFIRVQPIGGFVFLSRGGLDGKWLAVTNDGKLEEDWKDPEKRKNLKKLGGIMRTPVFVNKGRRVIVPFHNMGGGTKFHISDDGGLTWHVSRNGVTSPRHEARPPHQGVRWFNNAVEATVLEMKDGTLWALARTSQDQAWQAFSKDYGETWSKPEPSRFFGTLTMNTLGRLDDGTIVSLWTNTMALPENATAGNGTWEDVFTNRDSHHIAMSGDEGKTWYGFREIILDEHRNHPGYATLDGPEDRGKHQSEMVQLDKNRILISLGQHKNHRRLVIVDRRWVGAKTRATQTGKDLDSQWTIHTYIPQKKGHCSYNRKPSAELVQDPSGGTKKVLQIKRLDDPELVNEKSNVDYRNGGATWNFPNGTTGLVKFRFRVVDGEQADDSGLQVSLTDRLFNACDSTTKDYALFTFPIRLKPAPHLLLGMKKVPFTPGAWHEISLLWQGGQAVVSLDGKKAGTLKMANKSPNGASYIHFISTGSQPDAGILLDTVNARVK
SEQ ID NO:6-唾液酸酶,Am1757-具有另外的N末端甲硫氨酸和C末端接头+His6标签(加粗并加有下划线)
QQKAAPVPEPEVVATPPADAGRGLIRVDSREIRHYSGTRKEPDYLVSRDNGKTWEMKAAPAGYPPNYGGIPKESPAIVRNPLTREFIRVQPIGGFVFLSRGGLDGKWLAVTNDGKLEEDWKDPEKRKNLKKLGGIMRTPVFVNKGRRVIVPFHNMGGGTKFHISDDGGLTWHVSRNGVTSPRHEARPPHQGVRWFNNAVEATVLEMKDGTLWALARTSQDQAWQAFSKDYGETWSKPEPSRFFGTLTMNTLGRLDDGTIVSLWTNTMALPENATAGNGTWEDVFTNRDSHHIAMSGDEGKTWYGFREIILDEHRNHPGYATLDGPEDRGKHQSEMVQLDKNRILISLGQHKNHRRLVIVDRRWVGAKTRATQTGKDLDSQWTIHTYIPQKKGHCSYNRKPSAELVQDPSGGTKKVLQIKRLDDPELVNEKSNVDYRNGGATWNFPNGTTGLVKFRFRVVDGEQADDSGLQVSLTDRLFNACDSTTKDYALFTFPIRLKPAPHLLLGMKKVPFTPGAWHEISLLWQGGQAVVSLDGKKAGTLKMANKSPNGASYIHFISTGSQPDAGILLDTVNARVK/>
SEQ ID NO:7–来自口腔链球菌的O-糖苷酶
野生型(信号序列加有下划线;LPXTG细胞壁锚定基序的C末端元件加粗并加有下划线)
MDKRFFEKRCKFSIRKFTLGVASVMIGATFFAASPVLADQARVGSTDNLPSELADLDKKASDEGHDFDKEAAAQNPGSAETTEGPQTEEELLAQEKEKSEKPSNLPKELEDKLEKAEDNGREVDKDQLAQDTGKLVPEDVAKTTNGELNYGATVKIKTPSGEGSGIVVAKDLVLTVSHNFIKDSQEGNIRKVVDNDQGDGDIYSISYPGLPDVKFSKKDIIHWDREGYLKGFKNDLALVRLRTVLENTPVEVTKKPVVKKIGDKLHVFGYPEGKLNPIVNTTVDFAEPYGEGVQGIGYQGGKPGASGGGIFDTEGKLVGVHQNGVVGKRSGGILFSPAQLKWIQDHMQGISSVKPADLEEKEKPAEEKPKEDKPAAAKPETPKAVTPEWQTVANKEQQGTVTIREEKGVRYNQLSSTAQNDNDGKPALFEKQGLTVDANGNATVDLTFKDDSEKGKSRFGVFLKFKDTKNNVFVGYDQGGWFWEYKTPGNSTWYKGNRVAAPEPGSVNRLSITLKSDGQLNASNNDVNLFDTVTLPGAVNENLKNEKKILLKAGTYSNDRTVVSVKTDNQEGVKADDTPAQKETGPAVDDSKVTYDTIQSKVLKAVIDQAFPRVKEYTLNGHTLPGQVQQFNQVFINNHRITPEVTYKKINETTAEYLMKLRDDAHLINAEMTVRLQVVDNQLHFDVTKIVNHNQVTPGQKIDDERKLLSTISFLGNALVSVSSDQAGAKFDGATMSNNTHVSGDDHIDVTNPMKDLAKGYMYGFVSTDKLAAGVWSNSQNSYGGGSNDWTRLTAYKETVGNANYVGIHSSEWQWEKAYKGIVFPEYTKELPSAKVVITEDANADNKVDWQDGAIAYRSIMNNPQGWEKVKDITAYRIAMNFGSQAQNPFLMTLDGIKKINLHTDGLGQGVLLKGYGSEGHDSGHLNYADIGKRIGGVEDFKTLIEKAKKYGAHLGIHVNASETYPESKYFNENILRKNPDGSYSYGWNWLDQGINIDAAYDLAHGRLARWEDLKKKLGEGLDFIYVDVWGNGQSGDNGAWATHVLAKEINKQGWRFAIEWGHGGEYDSTFQHWAADLTYGGYTNKGINSAITRFIRNHQKDSWVGDYRSYGGAANYPLLGGYSMKDFEGWQGRSDYNGYVTNLFAHDVMTKYFQHFTVSKWENGTPVTMTDNGSTYKWTPEMKVELVDAAGNKVVVTRKSNDVNSPQYRERTVTLNGRVIQDGSAYLTPWNWDANGKKLPTEKEKMYYFNTQAGATTWTLPSDWANSKVYLYKLTDQGKTEEQELTVTDGKITLDLLANQPYVLYRSKQTNPEMSWSEGMHIYDQGFNSGTLKHWTISGDASKAEIVKSQGANEMLRIQGNKSKVSLTQKLTGLKPNTKYAVYVGVDNRSNAKASITVNTGEKEVTTYTNKSLALNYIKAYAHNNRRENATVDDTSYFQNMYAFFTTGSDVSNVTLTLSREAGDEATYFDEIRTFENNSSMYGDKHDTGQGTFKQDFENVAQGIFPFVVGGVEGVEDNRTHLSEKHDPYTQRGWNGKKVDDVIEGNWSLKTNGLVSRRNLVYQTIPQNFRFEAGKTYRVTFEYEAGSDNTYAFVVGKGEFQSGRRGTQASNLEMHELPNTWTDSKKAKKVTFLVTGAETGDTWVGIYSTGNASNTRGDAGGNANFRGYNDFMMDNLQIEEITLTGKMLTENALKNYLPTVAMTNYTKESMDALKEAVFNLSQADDDISVEEARAEIAKIEALKNALVQKKTALVAEDFESLDAPAQPGEGLENAFDGNVSSLWHTSWNGGDVGKPATMVLKEPTEITGLRYVPRASDSNGNLRDVKLVVTDESGKEHTFNVTDWPNNNKPKDIDFGKTIKAKKIVLTGTKTYGDGGDKYQSAAELIFTRPQVAETPLDLSGYEAALAKAQKLTDKDNQEEVASVQASMKYATDNHLLTERMVAYFADYLNQLKDSATKPDAPTSSKGEEQPPVLDVPEFKGGVNATEAAVHEVPEFKGGVNAVQALVHELPEYKGGANAVLAAANEVPEYKGGANAVEALVNEKPAYTGVLATAGDQAAPTVEKPEYPLTPSPVADTKTPGAKDEEKLPA
SEQ ID NO:8-来自口腔链球菌的O-糖苷酶
去除信号序列的野生型(LPXTG细胞壁锚定基序的C末端元件加粗并加有下划线)
DQARVGSTDNLPSELADLDKKASDEGHDFDKEAAAQNPGSAETTEGPQTEEELLAQEKEKSEKPSNLPKELEDKLEKAEDNGREVDKDQLAQDTGKLVPEDVAKTTNGELNYGATVKIKTPSGEGSGIVVAKDLVLTVSHNFIKDSQEGNIRKVVDNDQGDGDIYSISYPGLPDVKFSKKDIIHWDREGYLKGFKNDLALVRLRTVLENTPVEVTKKPVVKKIGDKLHVFGYPEGKLNPIVNTTVDFAEPYGEGVQGIGYQGGKPGASGGGIFDTEGKLVGVHQNGVVGKRSGGILFSPAQLKWIQDHMQGISSVKPADLEEKEKPAEEKPKEDKPAAAKPETPKAVTPEWQTVANKEQQGTVTIREEKGVRYNQLSSTAQNDNDGKPALFEKQGLTVDANGNATVDLTFKDDSEKGKSRFGVFLKFKDTKNNVFVGYDQGGWFWEYKTPGNSTWYKGNRVAAPEPGSVNRLSITLKSDGQLNASNNDVNLFDTVTLPGAVNENLKNEKKILLKAGTYSNDRTVVSVKTDNQEGVKADDTPAQKETGPAVDDSKVTYDTIQSKVLKAVIDQAFPRVKEYTLNGHTLPGQVQQFNQVFINNHRITPEVTYKKINETTAEYLMKLRDDAHLINAEMTVRLQVVDNQLHFDVTKIVNHNQVTPGQKIDDERKLLSTISFLGNALVSVSSDQAGAKFDGATMSNNTHVSGDDHIDVTNPMKDLAKGYMYGFVSTDKLAAGVWSNSQNSYGGGSNDWTRLTAYKETVGNANYVGIHSSEWQWEKAYKGIVFPEYTKELPSAKVVITEDANADNKVDWQDGAIAYRSIMNNPQGWEKVKDITAYRIAMNFGSQAQNPFLMTLDGIKKINLHTDGLGQGVLLKGYGSEGHDSGHLNYADIGKRIGGVEDFKTLIEKAKKYGAHLGIHVNASETYPESKYFNENILRKNPDGSYSYGWNWLDQGINIDAAYDLAHGRLARWEDLKKKLGEGLDFIYVDVWGNGQSGDNGAWATHVLAKEINKQGWRFAIEWGHGGEYDSTFQHWAADLTYGGYTNKGINSAITRFIRNHQKDSWVGDYRSYGGAANYPLLGGYSMKDFEGWQGRSDYNGYVTNLFAHDVMTKYFQHFTVSKWENGTPVTMTDNGSTYKWTPEMKVELVDAAGNKVVVTRKSNDVNSPQYRERTVTLNGRVIQDGSAYLTPWNWDANGKKLPTEKEKMYYFNTQAGATTWTLPSDWANSKVYLYKLTDQGKTEEQELTVTDGKITLDLLANQPYVLYRSKQTNPEMSWSEGMHIYDQGFNSGTLKHWTISGDASKAEIVKSQGANEMLRIQGNKSKVSLTQKLTGLKPNTKYAVYVGVDNRSNAKASITVNTGEKEVTTYTNKSLALNYIKAYAHNNRRENATVDDTSYFQNMYAFFTTGSDVSNVTLTLSREAGDEATYFDEIRTFENNSSMYGDKHDTGQGTFKQDFENVAQGIFPFVVGGVEGVEDNRTHLSEKHDPYTQRGWNGKKVDDVIEGNWSLKTNGLVSRRNLVYQTIPQNFRFEAGKTYRVTFEYEAGSDNTYAFVVGKGEFQSGRRGTQASNLEMHELPNTWTDSKKAKKVTFLVTGAETGDTWVGIYSTGNASNTRGDAGGNANFRGYNDFMMDNLQIEEITLTGKMLTENALKNYLPTVAMTNYTKESMDALKEAVFNLSQADDDISVEEARAEIAKIEALKNALVQKKTALVAEDFESLDAPAQPGEGLENAFDGNVSSLWHTSWNGGDVGKPATMVLKEPTEITGLRYVPRASDSNGNLRDVKLVVTDESGKEHTFNVTDWPNNNKPKDIDFGKTIKAKKIVLTGTKTYGDGGDKYQSAAELIFTRPQVAETPLDLSGYEAALAKAQKLTDKDNQEEVASVQASMKYATDNHLLTERMVAYFADYLNQLKDSATKPDAPTSSKGEEQPPVLDVPEFKGGVNATEAAVHEVPEFKGGVNAVQALVHELPEYKGGANAVLAAANEVPEYKGGANAVEALVNEKPAYTGVLATAGDQAAPTVEKPEYPLTPSPVADTKTPGAKDEEKLPA
SEQ ID NO:9-来自口腔链球菌的O-糖苷酶
去除信号序列和LPXTG细胞壁锚定基序的C末端元件的野生型
DQARVGSTDNLPSELADLDKKASDEGHDFDKEAAAQNPGSAETTEGPQTEEELLAQEKEKSEKPSNLPKELEDKLEKAEDNGREVDKDQLAQDTGKLVPEDVAKTTNGELNYGATVKIKTPSGEGSGIVVAKDLVLTVSHNFIKDSQEGNIRKVVDNDQGDGDIYSISYPGLPDVKFSKKDIIHWDREGYLKGFKNDLALVRLRTVLENTPVEVTKKPVVKKIGDKLHVFGYPEGKLNPIVNTTVDFAEPYGEGVQGIGYQGGKPGASGGGIFDTEGKLVGVHQNGVVGKRSGGILFSPAQLKWIQDHMQGISSVKPADLEEKEKPAEEKPKEDKPAAAKPETPKAVTPEWQTVANKEQQGTVTIREEKGVRYNQLSSTAQNDNDGKPALFEKQGLTVDANGNATVDLTFKDDSEKGKSRFGVFLKFKDTKNNVFVGYDQGGWFWEYKTPGNSTWYKGNRVAAPEPGSVNRLSITLKSDGQLNASNNDVNLFDTVTLPGAVNENLKNEKKILLKAGTYSNDRTVVSVKTDNQEGVKADDTPAQKETGPAVDDSKVTYDTIQSKVLKAVIDQAFPRVKEYTLNGHTLPGQVQQFNQVFINNHRITPEVTYKKINETTAEYLMKLRDDAHLINAEMTVRLQVVDNQLHFDVTKIVNHNQVTPGQKIDDERKLLSTISFLGNALVSVSSDQAGAKFDGATMSNNTHVSGDDHIDVTNPMKDLAKGYMYGFVSTDKLAAGVWSNSQNSYGGGSNDWTRLTAYKETVGNANYVGIHSSEWQWEKAYKGIVFPEYTKELPSAKVVITEDANADNKVDWQDGAIAYRSIMNNPQGWEKVKDITAYRIAMNFGSQAQNPFLMTLDGIKKINLHTDGLGQGVLLKGYGSEGHDSGHLNYADIGKRIGGVEDFKTLIEKAKKYGAHLGIHVNASETYPESKYFNENILRKNPDGSYSYGWNWLDQGINIDAAYDLAHGRLARWEDLKKKLGEGLDFIYVDVWGNGQSGDNGAWATHVLAKEINKQGWRFAIEWGHGGEYDSTFQHWAADLTYGGYTNKGINSAITRFIRNHQKDSWVGDYRSYGGAANYPLLGGYSMKDFEGWQGRSDYNGYVTNLFAHDVMTKYFQHFTVSKWENGTPVTMTDNGSTYKWTPEMKVELVDAAGNKVVVTRKSNDVNSPQYRERTVTLNGRVIQDGSAYLTPWNWDANGKKLPTEKEKMYYFNTQAGATTWTLPSDWANSKVYLYKLTDQGKTEEQELTVTDGKITLDLLANQPYVLYRSKQTNPEMSWSEGMHIYDQGFNSGTLKHWTISGDASKAEIVKSQGANEMLRIQGNKSKVSLTQKLTGLKPNTKYAVYVGVDNRSNAKASITVNTGEKEVTTYTNKSLALNYIKAYAHNNRRENATVDDTSYFQNMYAFFTTGSDVSNVTLTLSREAGDEATYFDEIRTFENNSSMYGDKHDTGQGTFKQDFENVAQGIFPFVVGGVEGVEDNRTHLSEKHDPYTQRGWNGKKVDDVIEGNWSLKTNGLVSRRNLVYQTIPQNFRFEAGKTYRVTFEYEAGSDNTYAFVVGKGEFQSGRRGTQASNLEMHELPNTWTDSKKAKKVTFLVTGAETGDTWVGIYSTGNASNTRGDAGGNANFRGYNDFMMDNLQIEEITLTGKMLTENALKNYLPTVAMTNYTKESMDALKEAVFNLSQADDDISVEEARAEIAKIEALKNALVQKKTALVAEDFESLDAPAQPGEGLENAFDGNVSSLWHTSWNGGDVGKPATMVLKEPTEITGLRYVPRASDSNGNLRDVKLVVTDESGKEHTFNVTDWPNNNKPKDIDFGKTIKAKKIVLTGTKTYGDGGDKYQSAAELIFTRPQVAETPLDLSGYEAALAKAQKLTDKDNQEEVASVQASMKYATDNHLLTERMVAYFADYLNQLKDSATKPDAPTSSKGEEQPPVLDVPEFKGGVNATEAAVHEVPEFKGGVNAVQALVHELPEYKGGANAVLAAANEVPEYKGGANAVEALVNEKPAYTGVLATAGDQAAPTVEKPEYPLTPSPVADTKTPGAKDEEKLPA
SEQ ID NO:10-来自口腔链球菌的O-糖苷酶,该O-糖苷酶具有另外的N末端甲硫氨酸和C末端GSGLE-His6标签(加粗并加有下划线)并且去除信号序列和LPXTG细胞壁锚定基序的C末端元件
DQARVGSTDNLPSELADLDKKASDEGHDFDKEAAAQNPGSAETTEGPQTEEELLAQEKEKSEKPSNLPKELEDKLEKAEDNGREVDKDQLAQDTGKLVPEDVAKTTNGELNYGATVKIKTPSGEGSGIVVAKDLVLTVSHNFIKDSQEGNIRKVVDNDQGDGDIYSISYPGLPDVKFSKKDIIHWDREGYLKGFKNDLALVRLRTVLENTPVEVTKKPVVKKIGDKLHVFGYPEGKLNPIVNTTVDFAEPYGEGVQGIGYQGGKPGASGGGIFDTEGKLVGVHQNGVVGKRSGGILFSPAQLKWIQDHMQGISSVKPADLEEKEKPAEEKPKEDKPAAAKPETPKAVTPEWQTVANKEQQGTVTIREEKGVRYNQLSSTAQNDNDGKPALFEKQGLTVDANGNATVDLTFKDDSEKGKSRFGVFLKFKDTKNNVFVGYDQGGWFWEYKTPGNSTWYKGNRVAAPEPGSVNRLSITLKSDGQLNASNNDVNLFDTVTLPGAVNENLKNEKKILLKAGTYSNDRTVVSVKTDNQEGVKADDTPAQKETGPAVDDSKVTYDTIQSKVLKAVIDQAFPRVKEYTLNGHTLPGQVQQFNQVFINNHRITPEVTYKKINETTAEYLMKLRDDAHLINAEMTVRLQVVDNQLHFDVTKIVNHNQVTPGQKIDDERKLLSTISFLGNALVSVSSDQAGAKFDGATMSNNTHVSGDDHIDVTNPMKDLAKGYMYGFVSTDKLAAGVWSNSQNSYGGGSNDWTRLTAYKETVGNANYVGIHSSEWQWEKAYKGIVFPEYTKELPSAKVVITEDANADNKVDWQDGAIAYRSIMNNPQGWEKVKDITAYRIAMNFGSQAQNPFLMTLDGIKKINLHTDGLGQGVLLKGYGSEGHDSGHLNYADIGKRIGGVEDFKTLIEKAKKYGAHLGIHVNASETYPESKYFNENILRKNPDGSYSYGWNWLDQGINIDAAYDLAHGRLARWEDLKKKLGEGLDFIYVDVWGNGQSGDNGAWATHVLAKEINKQGWRFAIEWGHGGEYDSTFQHWAADLTYGGYTNKGINSAITRFIRNHQKDSWVGDYRSYGGAANYPLLGGYSMKDFEGWQGRSDYNGYVTNLFAHDVMTKYFQHFTVSKWENGTPVTMTDNGSTYKWTPEMKVELVDAAGNKVVVTRKSNDVNSPQYRERTVTLNGRVIQDGSAYLTPWNWDANGKKLPTEKEKMYYFNTQAGATTWTLPSDWANSKVYLYKLTDQGKTEEQELTVTDGKITLDLLANQPYVLYRSKQTNPEMSWSEGMHIYDQGFNSGTLKHWTISGDASKAEIVKSQGANEMLRIQGNKSKVSLTQKLTGLKPNTKYAVYVGVDNRSNAKASITVNTGEKEVTTYTNKSLALNYIKAYAHNNRRENATVDDTSYFQNMYAFFTTGSDVSNVTLTLSREAGDEATYFDEIRTFENNSSMYGDKHDTGQGTFKQDFENVAQGIFPFVVGGVEGVEDNRTHLSEKHDPYTQRGWNGKKVDDVIEGNWSLKTNGLVSRRNLVYQTIPQNFRFEAGKTYRVTFEYEAGSDNTYAFVVGKGEFQSGRRGTQASNLEMHELPNTWTDSKKAKKVTFLVTGAETGDTWVGIYSTGNASNTRGDAGGNANFRGYNDFMMDNLQIEEITLTGKMLTENALKNYLPTVAMTNYTKESMDALKEAVFNLSQADDDISVEEARAEIAKIEALKNALVQKKTALVAEDFESLDAPAQPGEGLENAFDGNVSSLWHTSWNGGDVGKPATMVLKEPTEITGLRYVPRASDSNGNLRDVKLVVTDESGKEHTFNVTDWPNNNKPKDIDFGKTIKAKKIVLTGTKTYGDGGDKYQSAAELIFTRPQVAETPLDLSGYEAALAKAQKLTDKDNQEEVASVQASMKYATDNHLLTERMVAYFADYLNQLKDSATKPDAPTSSKGEEQPPVLDVPEFKGGVNATEAAVHEVPEFKGGVNAVQALVHELPEYKGGANAVLAAANEVPEYKGGANAVEALVNEKPAYTGVLATAGDQAAPTVEKPEYPLTPSPVADTKTPGAKDEEKLPA/>
SEQ ID NO:11-O-糖蛋白特异性内切蛋白酶
EVTVPDALKDRIALKKTARQLNIVYFLGSDTEPVPDYERRLSELLLYLQQFYGKEMQRHGYGARSFGLDIKSPGRVNIIEYKAKNPAAHYPYENGGGWKAAQELDEFFKAHPDRKKSQHTLIIMPTWNDEKNGPDNPGGVPFYGMGRNCFALDYPAFDIKHLGQKTREGRLLTKWYGGMAHELGHGLNLPHNHQTASDGKKYGTALMGSGNYTFGTSPTFLTPASCALLDACEVFSVTPSQQFYEGKPEVEVGDVAISFKGDQILVSGNYKSPQTVKALNVYIQDPPYAVNQDYDAVSFSRRLGKKSGKFSMKIDKKELEGLNNNEFRISLMFILANGLHMQKHFTFHWDALQDYRDGSKS
SEQ ID NO:12-O-糖蛋白特异性内切蛋白酶(LS)
MEVTVPDALKDRIALKKTARQLNIVYFLGSDTEPVPDYERRLSELLLYLQQFYGKEMQRHGYGARSFGLDIKSPGRVNIIEYKAKNPAAHYPYENGGGWKAAQELDEFFKAHPDRKKSQHTLIIMPTWNDEKNGPDNPGGVPFYGMGRNCFALDYPAFDIKHLGQKTREGRLLTKWYGGMAHELGHGLNLPHNHQTASDGKKYGTALMGSGNYTFGTSPTFLTPASCALLDACEVFSVTPSQQFYEGKPEVEVGDVAISFKGDQILVSGNYKSPQTVKALNVYIQDPPYAVNQDYDAVSFSRRLGKKSGKFSMKIDKKELEGLNNNEFRISLMFILANGLHMQKHFTFHWDALQDYRDGSKSGSGHHHHHH
SEQ ID NO:13–编码SEQ ID NO:10
ATGGACCAAGCGCGTGTGGGTAGCACCGATAACCTGCCGAGCGAGCTGGCGGATCTGGACAAGAAAGCGAGCGACGAAGGCCACGATTTTGACAAAGAGGCGGCGGCGCAGAACCCGGGTAGCGCGGAAACCACCGAAGGTCCGCAGACCGAGGAAGAGCTGCTGGCGCAAGAAAAAGAGAAGAGCGAGAAGCCGAGCAACCTGCCGAAAGAACTGGAGGATAAACTGGAAAAGGCGGAGGACAACGGTCGTGAAGTGGATAAAGACCAGCTGGCGCAAGACACCGGCAAGCTGGTGCCGGAGGATGTTGCGAAAACCACCAACGGTGAACTGAACTACGGCGCGACCGTTAAAATTAAGACCCCGAGCGGCGAGGGTAGCGGTATTGTGGTTGCGAAGGACCTGGTGCTGACCGTTAGCCACAACTTCATTAAGGATAGCCAGGAAGGTAATATCCGTAAAGTGGTTGATAACGACCAAGGCGATGGTGACATCTACAGCATTAGCTATCCGGGCCTGCCGGACGTTAAGTTCAGCAAGAAAGATATCATCCACTGGGACCGTGAGGGTTACCTGAAAGGCTTCAAGAACGATCTGGCGCTGGTGCGTCTGCGTACCGTTCTGGAAAACACCCCGGTTGAGGTGACCAAGAAACCGGTGGTTAAGAAAATTGGTGACAAGCTGCACGTGTTTGGTTATCCGGAGGGCAAACTGAACCCGATCGTGAACACCACCGTTGATTTCGCGGAACCGTACGGCGAGGGTGTTCAGGGCATTGGTTATCAAGGTGGCAAACCGGGCGCGAGCGGTGGCGGTATCTTTGACACCGAAGGCAAGCTGGTTGGCGTGCACCAGAACGGTGTGGTTGGCAAACGTAGCGGCGGTATTCTGTTCAGCCCGGCGCAACTGAAGTGGATTCAGGACCACATGCAAGGTATCAGCAGCGTGAAACCGGCGGATCTGGAAGAGAAAGAGAAGCCGGCGGAAGAGAAACCGAAGGAAGACAAGCCGGCGGCGGCGAAGCCGGAAACCCCGAAAGCGGTTACCCCGGAGTGGCAAACCGTGGCGAACAAGGAACAGCAAGGTACCGTTACCATCCGTGAAGAGAAAGGCGTGCGTTACAACCAGCTGAGCAGCACCGCGCAAAACGATAACGACGGCAAGCCGGCGCTGTTTGAGAAACAGGGTCTGACCGTTGACGCGAACGGCAACGCGACCGTGGATCTGACCTTCAAGGACGATAGCGAAAAAGGCAAGAGCCGTTTCGGCGTTTTTCTGAAATTCAAGGACACCAAAAACAACGTTTTTGTGGGTTACGATCAAGGCGGTTGGTTCTGGGAGTATAAGACCCCGGGTAACAGCACCTGGTACAAGGGTAACCGTGTGGCGGCGCCGGAACCGGGTAGCGTGAACCGTCTGAGCATTACCCTGAAAAGCGACGGCCAGCTGAACGCGAGCAACAACGATGTGAACCTGTTCGACACCGTTACCCTGCCGGGTGCGGTGAACGAAAACCTGAAGAACGAGAAGAAAATCCTGCTGAAAGCGGGCACCTACAGCAACGACCGTACCGTGGTTAGCGTTAAGACCGATAACCAGGAAGGTGTGAAAGCGGACGATACCCCGGCGCAAAAGGAAACCGGTCCGGCGGTGGACGATAGCAAGGTTACCTACGACACCATTCAGAGCAAAGTGCTGAAGGCGGTTATCGATCAAGCGTTTCCGCGTGTGAAAGAGTATACCCTGAACGGTCACACCCTGCCGGGTCAGGTTCAGCAATTTAACCAAGTGTTCATTAACAACCACCGTATCACCCCGGAAGTGACCTATAAGAAAATTAACGAAACCACCGCGGAGTACCTGATGAAGCTGCGTGACGATGCGCACCTGATCAACGCGGAAATGACCGTGCGTCTGCAGGTGGTTGATAACCAACTGCACTTCGACGTGACCAAAATTGTTAACCACAACCAGGTTACCCCGGGTCAAAAGATTGACGATGAGCGTAAACTGCTGAGCACCATCAGCTTTCTGGGCAACGCGCTGGTTAGCGTGAGCAGCGATCAAGCGGGTGCGAAGTTTGATGGTGCGACCATGAGCAACAACACCCACGTTAGCGGTGACGATCACATCGATGTGACCAACCCGATGAAAGACCTGGCGAAGGGTTACATGTATGGCTTTGTTAGCACCGACAAGCTGGCGGCGGGTGTGTGGAGCAACAGCCAAAACAGCTACGGCGGTGGCAGCAACGATTGGACCCGTCTGACCGCGTATAAAGAAACCGTTGGTAACGCGAACTACGTGGGCATTCACAGCAGCGAATGGCAGTGGGAGAAAGCGTACAAGGGTATCGTGTTCCCGGAATATACCAAGGAGCTGCCGAGCGCGAAAGTGGTTATCACCGAGGATGCGAACGCGGACAACAAAGTGGATTGGCAGGACGGTGCGATTGCGTACCGTAGCATCATGAACAACCCGCAAGGCTGGGAAAAAGTTAAGGACATTACCGCGTATCGTATCGCGATGAACTTTGGTAGCCAGGCGCAAAACCCGTTCCTGATGACCCTGGACGGCATCAAGAAAATTAACCTGCACACCGATGGCCTGGGTCAGGGCGTTCTGCTGAAGGGTTATGGTAGCGAGGGTCATGACAGCGGTCACCTGAACTACGCGGATATCGGTAAACGTATTGGTGGCGTGGAAGACTTTAAGACCCTGATTGAGAAAGCGAAGAAATACGGTGCGCACCTGGGCATCCACGTTAACGCGAGCGAAACCTACCCGGAGAGCAAGTATTTCAACGAAAACATTCTGCGTAAAAACCCGGACGGTAGCTACAGCTATGGCTGGAACTGGCTGGATCAGGGTATCAACATTGATGCGGCGTACGACCTGGCGCATGGCCGTCTGGCGCGTTGGGAGGACCTGAAGAAAAAGCTGGGTGAAGGCCTGGATTTTATCTATGTTGACGTGTGGGGTAACGGTCAGAGCGGTGATAACGGTGCGTGGGCGACCCATGTGCTGGCGAAAGAGATTAACAAGCAAGGTTGGCGTTTTGCGATCGAATGGGGCCACGGTGGCGAGTACGACAGCACCTTCCAGCACTGGGCGGCGGATCTGACCTACGGTGGCTATACCAACAAGGGTATCAACAGCGCGATTACCCGTTTCATCCGTAACCACCAGAAAGATAGCTGGGTTGGCGACTACCGTAGCTATGGTGGCGCGGCGAACTACCCGCTGCTGGGTGGCTATAGCATGAAGGACTTTGAGGGTTGGCAAGGCCGTAGCGATTACAACGGTTATGTTACCAACCTGTTCGCGCACGACGTGATGACCAAGTACTTTCAGCACTTCACCGTTAGCAAATGGGAAAACGGTACCCCGGTGACCATGACCGATAACGGCAGCACCTATAAGTGGACCCCGGAAATGAAAGTGGAGCTGGTTGACGCGGCGGGTAACAAGGTGGTTGTGACCCGTAAAAGCAACGATGTGAACAGCCCGCAGTACCGTGAGCGTACCGTTACCCTGAACGGTCGTGTGATCCAAGACGGCAGCGCGTATCTGACCCCGTGGAACTGGGATGCGAACGGTAAAAAGCTGCCGACCGAAAAAGAGAAGATGTACTATTTTAACACCCAAGCGGGTGCGACCACCTGGACCCTGCCGAGCGACTGGGCGAACAGCAAGGTTTACCTGTATAAACTGACCGATCAGGGCAAGACCGAGGAGCAAGAACTGACCGTGACCGATGGCAAAATTACCCTGGACCTGCTGGCGAACCAGCCGTACGTTCTGTATCGTAGCAAGCAAACCAACCCGGAAATGAGCTGGAGCGAGGGTATGCACATCTACGACCAAGGTTTCAACAGCGGCACCCTGAAACACTGGACCATTAGCGGCGATGCGAGCAAGGCGGAGATCGTGAAAAGCCAGGGTGCGAACGAAATGCTGCGTATCCAAGGCAACAAAAGCAAGGTTAGCCTGACCCAGAAGCTGACCGGTCTGAAACCGAACACCAAGTACGCGGTTTATGTGGGCGTTGACAACCGTAGCAACGCGAAAGCGAGCATTACCGTTAACACCGGTGAAAAAGAGGTGACCACCTACACCAACAAGAGCCTGGCGCTGAACTACATCAAAGCGTATGCGCACAACAACCGTCGTGAGAACGCGACCGTGGACGATACCAGCTACTTCCAGAACATGTATGCGTTCTTTACCACCGGTAGCGACGTGAGCAACGTTACCCTGACCCTGAGCCGTGAAGCGGGCGATGAGGCGACCTATTTTGACGAAATTCGTACCTTCGAGAACAACAGCAGCATGTACGGTGATAAGCACGACACCGGTCAGGGCACCTTTAAACAAGATTTCGAAAACGTTGCGCAAGGTATCTTCCCGTTTGTTGTGGGTGGCGTGGAAGGCGTTGAGGACAACCGTACCCACCTGAGCGAGAAGCACGATCCGTACACCCAGCGTGGTTGGAACGGCAAAAAGGTGGACGATGTTATTGAGGGTAACTGGAGCCTGAAAACCAACGGCCTGGTTAGCCGTCGTAACCTGGTGTACCAGACCATCCCGCAAAACTTCCGTTTTGAGGCGGGCAAGACCTACCGTGTGACCTTTGAATATGAGGCGGGCAGCGACAACACCTATGCGTTTGTTGTGGGTAAAGGCGAATTCCAGAGCGGTCGTCGTGGCACCCAAGCGAGCAACCTGGAAATGCACGAGCTGCCGAACACCTGGACCGATAGCAAAAAGGCGAAAAAGGTGACCTTCCTGGTTACCGGTGCGGAAACCGGTGACACCTGGGTGGGTATCTACAGCACCGGCAACGCGAGCAACACCCGTGGTGATGCGGGTGGCAACGCGAACTTTCGTGGCTATAACGATTTCATGATGGACAACCTGCAAATCGAAGAGATTACCCTGACCGGCAAGATGCTGACCGAAAACGCGCTGAAAAACTATCTGCCGACCGTTGCGATGACCAACTACACCAAGGAAAGCATGGACGCGCTGAAAGAGGCGGTTTTCAACCTGAGCCAGGCGGACGATGACATCAGCGTGGAAGAGGCGCGTGCGGAAATCGCGAAGATTGAGGCGCTGAAAAACGCGCTGGTTCAGAAAAAGACCGCGCTGGTTGCGGAAGATTTTGAGAGCCTGGATGCGCCGGCGCAACCGGGTGAAGGCCTGGAGAACGCGTTCGACGGTAACGTTAGCAGCCTGTGGCACACCAGCTGGAACGGTGGCGATGTTGGCAAGCCGGCGACCATGGTGCTGAAAGAACCGACCGAGATCACCGGTCTGCGTTATGTGCCGCGTGCGAGCGATAGCAACGGCAACCTGCGTGACGTTAAGCTGGTTGTGACCGATGAAAGCGGTAAAGAGCACACCTTTAACGTGACCGACTGGCCGAACAACAACAAACCGAAGGATATTGACTTCGGCAAAACCATTAAGGCGAAAAAGATCGTTCTGACCGGTACCAAGACCTACGGCGATGGTGGCGACAAATATCAGAGCGCGGCGGAGCTGATCTTTACCCGTCCGCAAGTGGCGGAAACCCCGCTGGATCTGAGCGGTTACGAAGCGGCGCTGGCGAAAGCGCAGAAGCTGACCGATAAGGACAACCAGGAAGAGGTGGCGAGCGTTCAAGCGAGCATGAAATATGCGACCGACAACCACCTGCTGACCGAACGTATGGTTGCGTACTTCGCGGATTATCTGAACCAACTGAAGGATAGCGCGACCAAACCGGATGCGCCGACCAGCAGCAAGGGTGAAGAACAGCCGCCGGTGCTGGATGTTCCGGAGTTTAAAGGTGGCGTGAACGCGACCGAGGCGGCGGTGCACGAAGTTCCGGAGTTCAAGGGTGGCGTGAACGCGGTTCAGGCGCTGGTTCACGAACTGCCGGAGTATAAAGGTGGCGCGAACGCGGTTCTGGCGGCGGCGAACGAAGTGCCGGAGTACAAGGGTGGCGCGAACGCGGTGGAAGCGCTGGTTAACGAGAAACCGGCGTATACCGGTGTTCTGGCGACCGCGGGCGACCAGGCGGCGCCGACCGTGGAAAAACCGGAGTACCCGCTGACCCCGAGCCCGGTTGCGGACACCAAAACCCCGGGTGCGAAAGATGAAGAGAAGCTGCCGGCGGGTAGCGGCCTCGAGCACCACCACCACCACCACTGA
序列表
<110> 杰诺维斯公司
<120> 用于聚糖分析的工具
<130> N410969WO
<150> GB1708471.6
<151> 2017-05-26
<150> GB1708476.5
<151> 2017-05-26
<150> GB1806655.5
<151> 2018-04-24
<160> 13
<170> PatentIn version 3.5
<210> 1
<211> 419
<212> PRT
<213> 嗜粘蛋白艾克曼菌
<400> 1
Met Thr Trp Leu Leu Cys Gly Arg Gly Lys Trp Asn Lys Val Lys Arg
1 5 10 15
Met Met Asn Ser Val Phe Lys Cys Leu Met Ser Ala Val Cys Ala Val
20 25 30
Ala Leu Pro Ala Phe Gly Gln Glu Glu Lys Thr Gly Phe Pro Thr Asp
35 40 45
Arg Ala Val Thr Val Phe Ser Ala Gly Glu Gly Asn Pro Tyr Ala Ser
50 55 60
Ile Arg Ile Pro Ala Leu Leu Ser Ile Gly Lys Gly Gln Leu Leu Ala
65 70 75 80
Phe Ala Glu Gly Arg Tyr Lys Asn Thr Asp Gln Gly Glu Asn Asp Ile
85 90 95
Ile Met Ser Val Ser Lys Asn Gly Gly Lys Thr Trp Ser Arg Pro Arg
100 105 110
Ala Ile Ala Lys Ala His Gly Ala Thr Phe Asn Asn Pro Cys Pro Val
115 120 125
Tyr Asp Ala Lys Thr Arg Thr Val Thr Val Val Phe Gln Arg Tyr Pro
130 135 140
Ala Gly Val Lys Glu Arg Gln Pro Asn Ile Pro Asp Gly Trp Asp Asp
145 150 155 160
Glu Lys Cys Ile Arg Asn Phe Met Ile Gln Ser Arg Asn Gly Gly Ser
165 170 175
Ser Trp Thr Lys Pro Gln Glu Ile Thr Lys Thr Thr Lys Arg Pro Ser
180 185 190
Gly Val Asp Ile Met Ala Ser Gly Pro Asn Ala Gly Thr Gln Leu Lys
195 200 205
Ser Gly Ala His Lys Gly Arg Leu Val Ile Pro Met Asn Glu Gly Pro
210 215 220
Phe Gly Lys Trp Val Ile Ser Cys Ile Tyr Ser Asp Asp Gly Gly Lys
225 230 235 240
Ser Trp Lys Leu Gly Gln Pro Thr Ala Asn Met Lys Gly Met Val Asn
245 250 255
Glu Thr Ser Ile Ala Glu Thr Asp Asn Gly Gly Val Val Met Val Ala
260 265 270
Arg His Trp Gly Ala Gly Asn Cys Arg Arg Ile Ala Trp Ser Gln Asp
275 280 285
Gly Gly Glu Thr Trp Gly Gln Val Glu Asp Ala Pro Glu Leu Phe Cys
290 295 300
Asp Ser Thr Gln Asn Ser Leu Met Thr Tyr Ser Leu Ser Asp Gln Pro
305 310 315 320
Ala Tyr Gly Gly Lys Ser Arg Ile Leu Phe Ser Gly Pro Ser Ala Gly
325 330 335
Arg Arg Ile Lys Gly Gln Val Ala Met Ser Tyr Asp Asn Gly Lys Thr
340 345 350
Trp Pro Val Lys Lys Leu Leu Gly Glu Gly Gly Phe Ala Tyr Ser Ser
355 360 365
Leu Ala Met Val Glu Pro Gly Ile Val Gly Val Leu Tyr Glu Glu Asn
370 375 380
Gln Glu His Ile Lys Lys Leu Lys Phe Val Pro Ile Thr Met Glu Trp
385 390 395 400
Leu Thr Asp Gly Glu Asp Thr Gly Leu Ala Pro Gly Lys Lys Ala Pro
405 410 415
Val Leu Lys
<210> 2
<211> 381
<212> PRT
<213> 嗜粘蛋白艾克曼菌
<400> 2
Gln Glu Glu Lys Thr Gly Phe Pro Thr Asp Arg Ala Val Thr Val Phe
1 5 10 15
Ser Ala Gly Glu Gly Asn Pro Tyr Ala Ser Ile Arg Ile Pro Ala Leu
20 25 30
Leu Ser Ile Gly Lys Gly Gln Leu Leu Ala Phe Ala Glu Gly Arg Tyr
35 40 45
Lys Asn Thr Asp Gln Gly Glu Asn Asp Ile Ile Met Ser Val Ser Lys
50 55 60
Asn Gly Gly Lys Thr Trp Ser Arg Pro Arg Ala Ile Ala Lys Ala His
65 70 75 80
Gly Ala Thr Phe Asn Asn Pro Cys Pro Val Tyr Asp Ala Lys Thr Arg
85 90 95
Thr Val Thr Val Val Phe Gln Arg Tyr Pro Ala Gly Val Lys Glu Arg
100 105 110
Gln Pro Asn Ile Pro Asp Gly Trp Asp Asp Glu Lys Cys Ile Arg Asn
115 120 125
Phe Met Ile Gln Ser Arg Asn Gly Gly Ser Ser Trp Thr Lys Pro Gln
130 135 140
Glu Ile Thr Lys Thr Thr Lys Arg Pro Ser Gly Val Asp Ile Met Ala
145 150 155 160
Ser Gly Pro Asn Ala Gly Thr Gln Leu Lys Ser Gly Ala His Lys Gly
165 170 175
Arg Leu Val Ile Pro Met Asn Glu Gly Pro Phe Gly Lys Trp Val Ile
180 185 190
Ser Cys Ile Tyr Ser Asp Asp Gly Gly Lys Ser Trp Lys Leu Gly Gln
195 200 205
Pro Thr Ala Asn Met Lys Gly Met Val Asn Glu Thr Ser Ile Ala Glu
210 215 220
Thr Asp Asn Gly Gly Val Val Met Val Ala Arg His Trp Gly Ala Gly
225 230 235 240
Asn Cys Arg Arg Ile Ala Trp Ser Gln Asp Gly Gly Glu Thr Trp Gly
245 250 255
Gln Val Glu Asp Ala Pro Glu Leu Phe Cys Asp Ser Thr Gln Asn Ser
260 265 270
Leu Met Thr Tyr Ser Leu Ser Asp Gln Pro Ala Tyr Gly Gly Lys Ser
275 280 285
Arg Ile Leu Phe Ser Gly Pro Ser Ala Gly Arg Arg Ile Lys Gly Gln
290 295 300
Val Ala Met Ser Tyr Asp Asn Gly Lys Thr Trp Pro Val Lys Lys Leu
305 310 315 320
Leu Gly Glu Gly Gly Phe Ala Tyr Ser Ser Leu Ala Met Val Glu Pro
325 330 335
Gly Ile Val Gly Val Leu Tyr Glu Glu Asn Gln Glu His Ile Lys Lys
340 345 350
Leu Lys Phe Val Pro Ile Thr Met Glu Trp Leu Thr Asp Gly Glu Asp
355 360 365
Thr Gly Leu Ala Pro Gly Lys Lys Ala Pro Val Leu Lys
370 375 380
<210> 3
<211> 393
<212> PRT
<213> 人工序列
<220>
<223> 唾液酸酶, Am0707 - 具有另外的N末端甲硫氨酸和C末端接头+His6标签
<400> 3
Met Gln Glu Glu Lys Thr Gly Phe Pro Thr Asp Arg Ala Val Thr Val
1 5 10 15
Phe Ser Ala Gly Glu Gly Asn Pro Tyr Ala Ser Ile Arg Ile Pro Ala
20 25 30
Leu Leu Ser Ile Gly Lys Gly Gln Leu Leu Ala Phe Ala Glu Gly Arg
35 40 45
Tyr Lys Asn Thr Asp Gln Gly Glu Asn Asp Ile Ile Met Ser Val Ser
50 55 60
Lys Asn Gly Gly Lys Thr Trp Ser Arg Pro Arg Ala Ile Ala Lys Ala
65 70 75 80
His Gly Ala Thr Phe Asn Asn Pro Cys Pro Val Tyr Asp Ala Lys Thr
85 90 95
Arg Thr Val Thr Val Val Phe Gln Arg Tyr Pro Ala Gly Val Lys Glu
100 105 110
Arg Gln Pro Asn Ile Pro Asp Gly Trp Asp Asp Glu Lys Cys Ile Arg
115 120 125
Asn Phe Met Ile Gln Ser Arg Asn Gly Gly Ser Ser Trp Thr Lys Pro
130 135 140
Gln Glu Ile Thr Lys Thr Thr Lys Arg Pro Ser Gly Val Asp Ile Met
145 150 155 160
Ala Ser Gly Pro Asn Ala Gly Thr Gln Leu Lys Ser Gly Ala His Lys
165 170 175
Gly Arg Leu Val Ile Pro Met Asn Glu Gly Pro Phe Gly Lys Trp Val
180 185 190
Ile Ser Cys Ile Tyr Ser Asp Asp Gly Gly Lys Ser Trp Lys Leu Gly
195 200 205
Gln Pro Thr Ala Asn Met Lys Gly Met Val Asn Glu Thr Ser Ile Ala
210 215 220
Glu Thr Asp Asn Gly Gly Val Val Met Val Ala Arg His Trp Gly Ala
225 230 235 240
Gly Asn Cys Arg Arg Ile Ala Trp Ser Gln Asp Gly Gly Glu Thr Trp
245 250 255
Gly Gln Val Glu Asp Ala Pro Glu Leu Phe Cys Asp Ser Thr Gln Asn
260 265 270
Ser Leu Met Thr Tyr Ser Leu Ser Asp Gln Pro Ala Tyr Gly Gly Lys
275 280 285
Ser Arg Ile Leu Phe Ser Gly Pro Ser Ala Gly Arg Arg Ile Lys Gly
290 295 300
Gln Val Ala Met Ser Tyr Asp Asn Gly Lys Thr Trp Pro Val Lys Lys
305 310 315 320
Leu Leu Gly Glu Gly Gly Phe Ala Tyr Ser Ser Leu Ala Met Val Glu
325 330 335
Pro Gly Ile Val Gly Val Leu Tyr Glu Glu Asn Gln Glu His Ile Lys
340 345 350
Lys Leu Lys Phe Val Pro Ile Thr Met Glu Trp Leu Thr Asp Gly Glu
355 360 365
Asp Thr Gly Leu Ala Pro Gly Lys Lys Ala Pro Val Leu Lys Gly Ser
370 375 380
Gly Leu Glu His His His His His His
385 390
<210> 4
<211> 595
<212> PRT
<213> 嗜粘蛋白艾克曼菌
<400> 4
Met Lys Asn Leu Leu Phe Ala Leu Leu Thr Gly Ser Phe Cys Cys Cys
1 5 10 15
Tyr Ala Gln Gln Lys Ala Ala Pro Val Pro Glu Pro Glu Val Val Ala
20 25 30
Thr Pro Pro Ala Asp Ala Gly Arg Gly Leu Ile Arg Val Asp Ser Arg
35 40 45
Glu Ile Arg His Tyr Ser Gly Thr Arg Lys Glu Pro Asp Tyr Leu Val
50 55 60
Ser Arg Asp Asn Gly Lys Thr Trp Glu Met Lys Ala Ala Pro Ala Gly
65 70 75 80
Tyr Pro Pro Asn Tyr Gly Gly Ile Pro Lys Glu Ser Pro Ala Ile Val
85 90 95
Arg Asn Pro Leu Thr Arg Glu Phe Ile Arg Val Gln Pro Ile Gly Gly
100 105 110
Phe Val Phe Leu Ser Arg Gly Gly Leu Asp Gly Lys Trp Leu Ala Val
115 120 125
Thr Asn Asp Gly Lys Leu Glu Glu Asp Trp Lys Asp Pro Glu Lys Arg
130 135 140
Lys Asn Leu Lys Lys Leu Gly Gly Ile Met Arg Thr Pro Val Phe Val
145 150 155 160
Asn Lys Gly Arg Arg Val Ile Val Pro Phe His Asn Met Gly Gly Gly
165 170 175
Thr Lys Phe His Ile Ser Asp Asp Gly Gly Leu Thr Trp His Val Ser
180 185 190
Arg Asn Gly Val Thr Ser Pro Arg His Glu Ala Arg Pro Pro His Gln
195 200 205
Gly Val Arg Trp Phe Asn Asn Ala Val Glu Ala Thr Val Leu Glu Met
210 215 220
Lys Asp Gly Thr Leu Trp Ala Leu Ala Arg Thr Ser Gln Asp Gln Ala
225 230 235 240
Trp Gln Ala Phe Ser Lys Asp Tyr Gly Glu Thr Trp Ser Lys Pro Glu
245 250 255
Pro Ser Arg Phe Phe Gly Thr Leu Thr Met Asn Thr Leu Gly Arg Leu
260 265 270
Asp Asp Gly Thr Ile Val Ser Leu Trp Thr Asn Thr Met Ala Leu Pro
275 280 285
Glu Asn Ala Thr Ala Gly Asn Gly Thr Trp Glu Asp Val Phe Thr Asn
290 295 300
Arg Asp Ser His His Ile Ala Met Ser Gly Asp Glu Gly Lys Thr Trp
305 310 315 320
Tyr Gly Phe Arg Glu Ile Ile Leu Asp Glu His Arg Asn His Pro Gly
325 330 335
Tyr Ala Thr Leu Asp Gly Pro Glu Asp Arg Gly Lys His Gln Ser Glu
340 345 350
Met Val Gln Leu Asp Lys Asn Arg Ile Leu Ile Ser Leu Gly Gln His
355 360 365
Lys Asn His Arg Arg Leu Val Ile Val Asp Arg Arg Trp Val Gly Ala
370 375 380
Lys Thr Arg Ala Thr Gln Thr Gly Lys Asp Leu Asp Ser Gln Trp Thr
385 390 395 400
Ile His Thr Tyr Ile Pro Gln Lys Lys Gly His Cys Ser Tyr Asn Arg
405 410 415
Lys Pro Ser Ala Glu Leu Val Gln Asp Pro Ser Gly Gly Thr Lys Lys
420 425 430
Val Leu Gln Ile Lys Arg Leu Asp Asp Pro Glu Leu Val Asn Glu Lys
435 440 445
Ser Asn Val Asp Tyr Arg Asn Gly Gly Ala Thr Trp Asn Phe Pro Asn
450 455 460
Gly Thr Thr Gly Leu Val Lys Phe Arg Phe Arg Val Val Asp Gly Glu
465 470 475 480
Gln Ala Asp Asp Ser Gly Leu Gln Val Ser Leu Thr Asp Arg Leu Phe
485 490 495
Asn Ala Cys Asp Ser Thr Thr Lys Asp Tyr Ala Leu Phe Thr Phe Pro
500 505 510
Ile Arg Leu Lys Pro Ala Pro His Leu Leu Leu Gly Met Lys Lys Val
515 520 525
Pro Phe Thr Pro Gly Ala Trp His Glu Ile Ser Leu Leu Trp Gln Gly
530 535 540
Gly Gln Ala Val Val Ser Leu Asp Gly Lys Lys Ala Gly Thr Leu Lys
545 550 555 560
Met Ala Asn Lys Ser Pro Asn Gly Ala Ser Tyr Ile His Phe Ile Ser
565 570 575
Thr Gly Ser Gln Pro Asp Ala Gly Ile Leu Leu Asp Thr Val Asn Ala
580 585 590
Arg Val Lys
595
<210> 5
<211> 577
<212> PRT
<213> 嗜粘蛋白艾克曼菌
<400> 5
Gln Gln Lys Ala Ala Pro Val Pro Glu Pro Glu Val Val Ala Thr Pro
1 5 10 15
Pro Ala Asp Ala Gly Arg Gly Leu Ile Arg Val Asp Ser Arg Glu Ile
20 25 30
Arg His Tyr Ser Gly Thr Arg Lys Glu Pro Asp Tyr Leu Val Ser Arg
35 40 45
Asp Asn Gly Lys Thr Trp Glu Met Lys Ala Ala Pro Ala Gly Tyr Pro
50 55 60
Pro Asn Tyr Gly Gly Ile Pro Lys Glu Ser Pro Ala Ile Val Arg Asn
65 70 75 80
Pro Leu Thr Arg Glu Phe Ile Arg Val Gln Pro Ile Gly Gly Phe Val
85 90 95
Phe Leu Ser Arg Gly Gly Leu Asp Gly Lys Trp Leu Ala Val Thr Asn
100 105 110
Asp Gly Lys Leu Glu Glu Asp Trp Lys Asp Pro Glu Lys Arg Lys Asn
115 120 125
Leu Lys Lys Leu Gly Gly Ile Met Arg Thr Pro Val Phe Val Asn Lys
130 135 140
Gly Arg Arg Val Ile Val Pro Phe His Asn Met Gly Gly Gly Thr Lys
145 150 155 160
Phe His Ile Ser Asp Asp Gly Gly Leu Thr Trp His Val Ser Arg Asn
165 170 175
Gly Val Thr Ser Pro Arg His Glu Ala Arg Pro Pro His Gln Gly Val
180 185 190
Arg Trp Phe Asn Asn Ala Val Glu Ala Thr Val Leu Glu Met Lys Asp
195 200 205
Gly Thr Leu Trp Ala Leu Ala Arg Thr Ser Gln Asp Gln Ala Trp Gln
210 215 220
Ala Phe Ser Lys Asp Tyr Gly Glu Thr Trp Ser Lys Pro Glu Pro Ser
225 230 235 240
Arg Phe Phe Gly Thr Leu Thr Met Asn Thr Leu Gly Arg Leu Asp Asp
245 250 255
Gly Thr Ile Val Ser Leu Trp Thr Asn Thr Met Ala Leu Pro Glu Asn
260 265 270
Ala Thr Ala Gly Asn Gly Thr Trp Glu Asp Val Phe Thr Asn Arg Asp
275 280 285
Ser His His Ile Ala Met Ser Gly Asp Glu Gly Lys Thr Trp Tyr Gly
290 295 300
Phe Arg Glu Ile Ile Leu Asp Glu His Arg Asn His Pro Gly Tyr Ala
305 310 315 320
Thr Leu Asp Gly Pro Glu Asp Arg Gly Lys His Gln Ser Glu Met Val
325 330 335
Gln Leu Asp Lys Asn Arg Ile Leu Ile Ser Leu Gly Gln His Lys Asn
340 345 350
His Arg Arg Leu Val Ile Val Asp Arg Arg Trp Val Gly Ala Lys Thr
355 360 365
Arg Ala Thr Gln Thr Gly Lys Asp Leu Asp Ser Gln Trp Thr Ile His
370 375 380
Thr Tyr Ile Pro Gln Lys Lys Gly His Cys Ser Tyr Asn Arg Lys Pro
385 390 395 400
Ser Ala Glu Leu Val Gln Asp Pro Ser Gly Gly Thr Lys Lys Val Leu
405 410 415
Gln Ile Lys Arg Leu Asp Asp Pro Glu Leu Val Asn Glu Lys Ser Asn
420 425 430
Val Asp Tyr Arg Asn Gly Gly Ala Thr Trp Asn Phe Pro Asn Gly Thr
435 440 445
Thr Gly Leu Val Lys Phe Arg Phe Arg Val Val Asp Gly Glu Gln Ala
450 455 460
Asp Asp Ser Gly Leu Gln Val Ser Leu Thr Asp Arg Leu Phe Asn Ala
465 470 475 480
Cys Asp Ser Thr Thr Lys Asp Tyr Ala Leu Phe Thr Phe Pro Ile Arg
485 490 495
Leu Lys Pro Ala Pro His Leu Leu Leu Gly Met Lys Lys Val Pro Phe
500 505 510
Thr Pro Gly Ala Trp His Glu Ile Ser Leu Leu Trp Gln Gly Gly Gln
515 520 525
Ala Val Val Ser Leu Asp Gly Lys Lys Ala Gly Thr Leu Lys Met Ala
530 535 540
Asn Lys Ser Pro Asn Gly Ala Ser Tyr Ile His Phe Ile Ser Thr Gly
545 550 555 560
Ser Gln Pro Asp Ala Gly Ile Leu Leu Asp Thr Val Asn Ala Arg Val
565 570 575
Lys
<210> 6
<211> 589
<212> PRT
<213> 人工序列
<220>
<223> 唾液酸酶, Am1757 - 具有另外的N末端甲硫氨酸和C末端接头+His6标签
<400> 6
Met Gln Gln Lys Ala Ala Pro Val Pro Glu Pro Glu Val Val Ala Thr
1 5 10 15
Pro Pro Ala Asp Ala Gly Arg Gly Leu Ile Arg Val Asp Ser Arg Glu
20 25 30
Ile Arg His Tyr Ser Gly Thr Arg Lys Glu Pro Asp Tyr Leu Val Ser
35 40 45
Arg Asp Asn Gly Lys Thr Trp Glu Met Lys Ala Ala Pro Ala Gly Tyr
50 55 60
Pro Pro Asn Tyr Gly Gly Ile Pro Lys Glu Ser Pro Ala Ile Val Arg
65 70 75 80
Asn Pro Leu Thr Arg Glu Phe Ile Arg Val Gln Pro Ile Gly Gly Phe
85 90 95
Val Phe Leu Ser Arg Gly Gly Leu Asp Gly Lys Trp Leu Ala Val Thr
100 105 110
Asn Asp Gly Lys Leu Glu Glu Asp Trp Lys Asp Pro Glu Lys Arg Lys
115 120 125
Asn Leu Lys Lys Leu Gly Gly Ile Met Arg Thr Pro Val Phe Val Asn
130 135 140
Lys Gly Arg Arg Val Ile Val Pro Phe His Asn Met Gly Gly Gly Thr
145 150 155 160
Lys Phe His Ile Ser Asp Asp Gly Gly Leu Thr Trp His Val Ser Arg
165 170 175
Asn Gly Val Thr Ser Pro Arg His Glu Ala Arg Pro Pro His Gln Gly
180 185 190
Val Arg Trp Phe Asn Asn Ala Val Glu Ala Thr Val Leu Glu Met Lys
195 200 205
Asp Gly Thr Leu Trp Ala Leu Ala Arg Thr Ser Gln Asp Gln Ala Trp
210 215 220
Gln Ala Phe Ser Lys Asp Tyr Gly Glu Thr Trp Ser Lys Pro Glu Pro
225 230 235 240
Ser Arg Phe Phe Gly Thr Leu Thr Met Asn Thr Leu Gly Arg Leu Asp
245 250 255
Asp Gly Thr Ile Val Ser Leu Trp Thr Asn Thr Met Ala Leu Pro Glu
260 265 270
Asn Ala Thr Ala Gly Asn Gly Thr Trp Glu Asp Val Phe Thr Asn Arg
275 280 285
Asp Ser His His Ile Ala Met Ser Gly Asp Glu Gly Lys Thr Trp Tyr
290 295 300
Gly Phe Arg Glu Ile Ile Leu Asp Glu His Arg Asn His Pro Gly Tyr
305 310 315 320
Ala Thr Leu Asp Gly Pro Glu Asp Arg Gly Lys His Gln Ser Glu Met
325 330 335
Val Gln Leu Asp Lys Asn Arg Ile Leu Ile Ser Leu Gly Gln His Lys
340 345 350
Asn His Arg Arg Leu Val Ile Val Asp Arg Arg Trp Val Gly Ala Lys
355 360 365
Thr Arg Ala Thr Gln Thr Gly Lys Asp Leu Asp Ser Gln Trp Thr Ile
370 375 380
His Thr Tyr Ile Pro Gln Lys Lys Gly His Cys Ser Tyr Asn Arg Lys
385 390 395 400
Pro Ser Ala Glu Leu Val Gln Asp Pro Ser Gly Gly Thr Lys Lys Val
405 410 415
Leu Gln Ile Lys Arg Leu Asp Asp Pro Glu Leu Val Asn Glu Lys Ser
420 425 430
Asn Val Asp Tyr Arg Asn Gly Gly Ala Thr Trp Asn Phe Pro Asn Gly
435 440 445
Thr Thr Gly Leu Val Lys Phe Arg Phe Arg Val Val Asp Gly Glu Gln
450 455 460
Ala Asp Asp Ser Gly Leu Gln Val Ser Leu Thr Asp Arg Leu Phe Asn
465 470 475 480
Ala Cys Asp Ser Thr Thr Lys Asp Tyr Ala Leu Phe Thr Phe Pro Ile
485 490 495
Arg Leu Lys Pro Ala Pro His Leu Leu Leu Gly Met Lys Lys Val Pro
500 505 510
Phe Thr Pro Gly Ala Trp His Glu Ile Ser Leu Leu Trp Gln Gly Gly
515 520 525
Gln Ala Val Val Ser Leu Asp Gly Lys Lys Ala Gly Thr Leu Lys Met
530 535 540
Ala Asn Lys Ser Pro Asn Gly Ala Ser Tyr Ile His Phe Ile Ser Thr
545 550 555 560
Gly Ser Gln Pro Asp Ala Gly Ile Leu Leu Asp Thr Val Asn Ala Arg
565 570 575
Val Lys Gly Ser Gly Leu Glu His His His His His His
580 585
<210> 7
<211> 2122
<212> PRT
<213> 口腔链球菌
<400> 7
Met Asp Lys Arg Phe Phe Glu Lys Arg Cys Lys Phe Ser Ile Arg Lys
1 5 10 15
Phe Thr Leu Gly Val Ala Ser Val Met Ile Gly Ala Thr Phe Phe Ala
20 25 30
Ala Ser Pro Val Leu Ala Asp Gln Ala Arg Val Gly Ser Thr Asp Asn
35 40 45
Leu Pro Ser Glu Leu Ala Asp Leu Asp Lys Lys Ala Ser Asp Glu Gly
50 55 60
His Asp Phe Asp Lys Glu Ala Ala Ala Gln Asn Pro Gly Ser Ala Glu
65 70 75 80
Thr Thr Glu Gly Pro Gln Thr Glu Glu Glu Leu Leu Ala Gln Glu Lys
85 90 95
Glu Lys Ser Glu Lys Pro Ser Asn Leu Pro Lys Glu Leu Glu Asp Lys
100 105 110
Leu Glu Lys Ala Glu Asp Asn Gly Arg Glu Val Asp Lys Asp Gln Leu
115 120 125
Ala Gln Asp Thr Gly Lys Leu Val Pro Glu Asp Val Ala Lys Thr Thr
130 135 140
Asn Gly Glu Leu Asn Tyr Gly Ala Thr Val Lys Ile Lys Thr Pro Ser
145 150 155 160
Gly Glu Gly Ser Gly Ile Val Val Ala Lys Asp Leu Val Leu Thr Val
165 170 175
Ser His Asn Phe Ile Lys Asp Ser Gln Glu Gly Asn Ile Arg Lys Val
180 185 190
Val Asp Asn Asp Gln Gly Asp Gly Asp Ile Tyr Ser Ile Ser Tyr Pro
195 200 205
Gly Leu Pro Asp Val Lys Phe Ser Lys Lys Asp Ile Ile His Trp Asp
210 215 220
Arg Glu Gly Tyr Leu Lys Gly Phe Lys Asn Asp Leu Ala Leu Val Arg
225 230 235 240
Leu Arg Thr Val Leu Glu Asn Thr Pro Val Glu Val Thr Lys Lys Pro
245 250 255
Val Val Lys Lys Ile Gly Asp Lys Leu His Val Phe Gly Tyr Pro Glu
260 265 270
Gly Lys Leu Asn Pro Ile Val Asn Thr Thr Val Asp Phe Ala Glu Pro
275 280 285
Tyr Gly Glu Gly Val Gln Gly Ile Gly Tyr Gln Gly Gly Lys Pro Gly
290 295 300
Ala Ser Gly Gly Gly Ile Phe Asp Thr Glu Gly Lys Leu Val Gly Val
305 310 315 320
His Gln Asn Gly Val Val Gly Lys Arg Ser Gly Gly Ile Leu Phe Ser
325 330 335
Pro Ala Gln Leu Lys Trp Ile Gln Asp His Met Gln Gly Ile Ser Ser
340 345 350
Val Lys Pro Ala Asp Leu Glu Glu Lys Glu Lys Pro Ala Glu Glu Lys
355 360 365
Pro Lys Glu Asp Lys Pro Ala Ala Ala Lys Pro Glu Thr Pro Lys Ala
370 375 380
Val Thr Pro Glu Trp Gln Thr Val Ala Asn Lys Glu Gln Gln Gly Thr
385 390 395 400
Val Thr Ile Arg Glu Glu Lys Gly Val Arg Tyr Asn Gln Leu Ser Ser
405 410 415
Thr Ala Gln Asn Asp Asn Asp Gly Lys Pro Ala Leu Phe Glu Lys Gln
420 425 430
Gly Leu Thr Val Asp Ala Asn Gly Asn Ala Thr Val Asp Leu Thr Phe
435 440 445
Lys Asp Asp Ser Glu Lys Gly Lys Ser Arg Phe Gly Val Phe Leu Lys
450 455 460
Phe Lys Asp Thr Lys Asn Asn Val Phe Val Gly Tyr Asp Gln Gly Gly
465 470 475 480
Trp Phe Trp Glu Tyr Lys Thr Pro Gly Asn Ser Thr Trp Tyr Lys Gly
485 490 495
Asn Arg Val Ala Ala Pro Glu Pro Gly Ser Val Asn Arg Leu Ser Ile
500 505 510
Thr Leu Lys Ser Asp Gly Gln Leu Asn Ala Ser Asn Asn Asp Val Asn
515 520 525
Leu Phe Asp Thr Val Thr Leu Pro Gly Ala Val Asn Glu Asn Leu Lys
530 535 540
Asn Glu Lys Lys Ile Leu Leu Lys Ala Gly Thr Tyr Ser Asn Asp Arg
545 550 555 560
Thr Val Val Ser Val Lys Thr Asp Asn Gln Glu Gly Val Lys Ala Asp
565 570 575
Asp Thr Pro Ala Gln Lys Glu Thr Gly Pro Ala Val Asp Asp Ser Lys
580 585 590
Val Thr Tyr Asp Thr Ile Gln Ser Lys Val Leu Lys Ala Val Ile Asp
595 600 605
Gln Ala Phe Pro Arg Val Lys Glu Tyr Thr Leu Asn Gly His Thr Leu
610 615 620
Pro Gly Gln Val Gln Gln Phe Asn Gln Val Phe Ile Asn Asn His Arg
625 630 635 640
Ile Thr Pro Glu Val Thr Tyr Lys Lys Ile Asn Glu Thr Thr Ala Glu
645 650 655
Tyr Leu Met Lys Leu Arg Asp Asp Ala His Leu Ile Asn Ala Glu Met
660 665 670
Thr Val Arg Leu Gln Val Val Asp Asn Gln Leu His Phe Asp Val Thr
675 680 685
Lys Ile Val Asn His Asn Gln Val Thr Pro Gly Gln Lys Ile Asp Asp
690 695 700
Glu Arg Lys Leu Leu Ser Thr Ile Ser Phe Leu Gly Asn Ala Leu Val
705 710 715 720
Ser Val Ser Ser Asp Gln Ala Gly Ala Lys Phe Asp Gly Ala Thr Met
725 730 735
Ser Asn Asn Thr His Val Ser Gly Asp Asp His Ile Asp Val Thr Asn
740 745 750
Pro Met Lys Asp Leu Ala Lys Gly Tyr Met Tyr Gly Phe Val Ser Thr
755 760 765
Asp Lys Leu Ala Ala Gly Val Trp Ser Asn Ser Gln Asn Ser Tyr Gly
770 775 780
Gly Gly Ser Asn Asp Trp Thr Arg Leu Thr Ala Tyr Lys Glu Thr Val
785 790 795 800
Gly Asn Ala Asn Tyr Val Gly Ile His Ser Ser Glu Trp Gln Trp Glu
805 810 815
Lys Ala Tyr Lys Gly Ile Val Phe Pro Glu Tyr Thr Lys Glu Leu Pro
820 825 830
Ser Ala Lys Val Val Ile Thr Glu Asp Ala Asn Ala Asp Asn Lys Val
835 840 845
Asp Trp Gln Asp Gly Ala Ile Ala Tyr Arg Ser Ile Met Asn Asn Pro
850 855 860
Gln Gly Trp Glu Lys Val Lys Asp Ile Thr Ala Tyr Arg Ile Ala Met
865 870 875 880
Asn Phe Gly Ser Gln Ala Gln Asn Pro Phe Leu Met Thr Leu Asp Gly
885 890 895
Ile Lys Lys Ile Asn Leu His Thr Asp Gly Leu Gly Gln Gly Val Leu
900 905 910
Leu Lys Gly Tyr Gly Ser Glu Gly His Asp Ser Gly His Leu Asn Tyr
915 920 925
Ala Asp Ile Gly Lys Arg Ile Gly Gly Val Glu Asp Phe Lys Thr Leu
930 935 940
Ile Glu Lys Ala Lys Lys Tyr Gly Ala His Leu Gly Ile His Val Asn
945 950 955 960
Ala Ser Glu Thr Tyr Pro Glu Ser Lys Tyr Phe Asn Glu Asn Ile Leu
965 970 975
Arg Lys Asn Pro Asp Gly Ser Tyr Ser Tyr Gly Trp Asn Trp Leu Asp
980 985 990
Gln Gly Ile Asn Ile Asp Ala Ala Tyr Asp Leu Ala His Gly Arg Leu
995 1000 1005
Ala Arg Trp Glu Asp Leu Lys Lys Lys Leu Gly Glu Gly Leu Asp
1010 1015 1020
Phe Ile Tyr Val Asp Val Trp Gly Asn Gly Gln Ser Gly Asp Asn
1025 1030 1035
Gly Ala Trp Ala Thr His Val Leu Ala Lys Glu Ile Asn Lys Gln
1040 1045 1050
Gly Trp Arg Phe Ala Ile Glu Trp Gly His Gly Gly Glu Tyr Asp
1055 1060 1065
Ser Thr Phe Gln His Trp Ala Ala Asp Leu Thr Tyr Gly Gly Tyr
1070 1075 1080
Thr Asn Lys Gly Ile Asn Ser Ala Ile Thr Arg Phe Ile Arg Asn
1085 1090 1095
His Gln Lys Asp Ser Trp Val Gly Asp Tyr Arg Ser Tyr Gly Gly
1100 1105 1110
Ala Ala Asn Tyr Pro Leu Leu Gly Gly Tyr Ser Met Lys Asp Phe
1115 1120 1125
Glu Gly Trp Gln Gly Arg Ser Asp Tyr Asn Gly Tyr Val Thr Asn
1130 1135 1140
Leu Phe Ala His Asp Val Met Thr Lys Tyr Phe Gln His Phe Thr
1145 1150 1155
Val Ser Lys Trp Glu Asn Gly Thr Pro Val Thr Met Thr Asp Asn
1160 1165 1170
Gly Ser Thr Tyr Lys Trp Thr Pro Glu Met Lys Val Glu Leu Val
1175 1180 1185
Asp Ala Ala Gly Asn Lys Val Val Val Thr Arg Lys Ser Asn Asp
1190 1195 1200
Val Asn Ser Pro Gln Tyr Arg Glu Arg Thr Val Thr Leu Asn Gly
1205 1210 1215
Arg Val Ile Gln Asp Gly Ser Ala Tyr Leu Thr Pro Trp Asn Trp
1220 1225 1230
Asp Ala Asn Gly Lys Lys Leu Pro Thr Glu Lys Glu Lys Met Tyr
1235 1240 1245
Tyr Phe Asn Thr Gln Ala Gly Ala Thr Thr Trp Thr Leu Pro Ser
1250 1255 1260
Asp Trp Ala Asn Ser Lys Val Tyr Leu Tyr Lys Leu Thr Asp Gln
1265 1270 1275
Gly Lys Thr Glu Glu Gln Glu Leu Thr Val Thr Asp Gly Lys Ile
1280 1285 1290
Thr Leu Asp Leu Leu Ala Asn Gln Pro Tyr Val Leu Tyr Arg Ser
1295 1300 1305
Lys Gln Thr Asn Pro Glu Met Ser Trp Ser Glu Gly Met His Ile
1310 1315 1320
Tyr Asp Gln Gly Phe Asn Ser Gly Thr Leu Lys His Trp Thr Ile
1325 1330 1335
Ser Gly Asp Ala Ser Lys Ala Glu Ile Val Lys Ser Gln Gly Ala
1340 1345 1350
Asn Glu Met Leu Arg Ile Gln Gly Asn Lys Ser Lys Val Ser Leu
1355 1360 1365
Thr Gln Lys Leu Thr Gly Leu Lys Pro Asn Thr Lys Tyr Ala Val
1370 1375 1380
Tyr Val Gly Val Asp Asn Arg Ser Asn Ala Lys Ala Ser Ile Thr
1385 1390 1395
Val Asn Thr Gly Glu Lys Glu Val Thr Thr Tyr Thr Asn Lys Ser
1400 1405 1410
Leu Ala Leu Asn Tyr Ile Lys Ala Tyr Ala His Asn Asn Arg Arg
1415 1420 1425
Glu Asn Ala Thr Val Asp Asp Thr Ser Tyr Phe Gln Asn Met Tyr
1430 1435 1440
Ala Phe Phe Thr Thr Gly Ser Asp Val Ser Asn Val Thr Leu Thr
1445 1450 1455
Leu Ser Arg Glu Ala Gly Asp Glu Ala Thr Tyr Phe Asp Glu Ile
1460 1465 1470
Arg Thr Phe Glu Asn Asn Ser Ser Met Tyr Gly Asp Lys His Asp
1475 1480 1485
Thr Gly Gln Gly Thr Phe Lys Gln Asp Phe Glu Asn Val Ala Gln
1490 1495 1500
Gly Ile Phe Pro Phe Val Val Gly Gly Val Glu Gly Val Glu Asp
1505 1510 1515
Asn Arg Thr His Leu Ser Glu Lys His Asp Pro Tyr Thr Gln Arg
1520 1525 1530
Gly Trp Asn Gly Lys Lys Val Asp Asp Val Ile Glu Gly Asn Trp
1535 1540 1545
Ser Leu Lys Thr Asn Gly Leu Val Ser Arg Arg Asn Leu Val Tyr
1550 1555 1560
Gln Thr Ile Pro Gln Asn Phe Arg Phe Glu Ala Gly Lys Thr Tyr
1565 1570 1575
Arg Val Thr Phe Glu Tyr Glu Ala Gly Ser Asp Asn Thr Tyr Ala
1580 1585 1590
Phe Val Val Gly Lys Gly Glu Phe Gln Ser Gly Arg Arg Gly Thr
1595 1600 1605
Gln Ala Ser Asn Leu Glu Met His Glu Leu Pro Asn Thr Trp Thr
1610 1615 1620
Asp Ser Lys Lys Ala Lys Lys Val Thr Phe Leu Val Thr Gly Ala
1625 1630 1635
Glu Thr Gly Asp Thr Trp Val Gly Ile Tyr Ser Thr Gly Asn Ala
1640 1645 1650
Ser Asn Thr Arg Gly Asp Ala Gly Gly Asn Ala Asn Phe Arg Gly
1655 1660 1665
Tyr Asn Asp Phe Met Met Asp Asn Leu Gln Ile Glu Glu Ile Thr
1670 1675 1680
Leu Thr Gly Lys Met Leu Thr Glu Asn Ala Leu Lys Asn Tyr Leu
1685 1690 1695
Pro Thr Val Ala Met Thr Asn Tyr Thr Lys Glu Ser Met Asp Ala
1700 1705 1710
Leu Lys Glu Ala Val Phe Asn Leu Ser Gln Ala Asp Asp Asp Ile
1715 1720 1725
Ser Val Glu Glu Ala Arg Ala Glu Ile Ala Lys Ile Glu Ala Leu
1730 1735 1740
Lys Asn Ala Leu Val Gln Lys Lys Thr Ala Leu Val Ala Glu Asp
1745 1750 1755
Phe Glu Ser Leu Asp Ala Pro Ala Gln Pro Gly Glu Gly Leu Glu
1760 1765 1770
Asn Ala Phe Asp Gly Asn Val Ser Ser Leu Trp His Thr Ser Trp
1775 1780 1785
Asn Gly Gly Asp Val Gly Lys Pro Ala Thr Met Val Leu Lys Glu
1790 1795 1800
Pro Thr Glu Ile Thr Gly Leu Arg Tyr Val Pro Arg Ala Ser Asp
1805 1810 1815
Ser Asn Gly Asn Leu Arg Asp Val Lys Leu Val Val Thr Asp Glu
1820 1825 1830
Ser Gly Lys Glu His Thr Phe Asn Val Thr Asp Trp Pro Asn Asn
1835 1840 1845
Asn Lys Pro Lys Asp Ile Asp Phe Gly Lys Thr Ile Lys Ala Lys
1850 1855 1860
Lys Ile Val Leu Thr Gly Thr Lys Thr Tyr Gly Asp Gly Gly Asp
1865 1870 1875
Lys Tyr Gln Ser Ala Ala Glu Leu Ile Phe Thr Arg Pro Gln Val
1880 1885 1890
Ala Glu Thr Pro Leu Asp Leu Ser Gly Tyr Glu Ala Ala Leu Ala
1895 1900 1905
Lys Ala Gln Lys Leu Thr Asp Lys Asp Asn Gln Glu Glu Val Ala
1910 1915 1920
Ser Val Gln Ala Ser Met Lys Tyr Ala Thr Asp Asn His Leu Leu
1925 1930 1935
Thr Glu Arg Met Val Ala Tyr Phe Ala Asp Tyr Leu Asn Gln Leu
1940 1945 1950
Lys Asp Ser Ala Thr Lys Pro Asp Ala Pro Thr Ser Ser Lys Gly
1955 1960 1965
Glu Glu Gln Pro Pro Val Leu Asp Val Pro Glu Phe Lys Gly Gly
1970 1975 1980
Val Asn Ala Thr Glu Ala Ala Val His Glu Val Pro Glu Phe Lys
1985 1990 1995
Gly Gly Val Asn Ala Val Gln Ala Leu Val His Glu Leu Pro Glu
2000 2005 2010
Tyr Lys Gly Gly Ala Asn Ala Val Leu Ala Ala Ala Asn Glu Val
2015 2020 2025
Pro Glu Tyr Lys Gly Gly Ala Asn Ala Val Glu Ala Leu Val Asn
2030 2035 2040
Glu Lys Pro Ala Tyr Thr Gly Val Leu Ala Thr Ala Gly Asp Gln
2045 2050 2055
Ala Ala Pro Thr Val Glu Lys Pro Glu Tyr Pro Leu Thr Pro Ser
2060 2065 2070
Pro Val Ala Asp Thr Lys Thr Pro Gly Ala Lys Asp Glu Glu Lys
2075 2080 2085
Leu Pro Ala Thr Gly Glu His Ser Ser Glu Val Ala Leu Phe Leu
2090 2095 2100
Ala Ser Val Ser Ile Ala Leu Ser Ala Ala Val Leu Ala Thr Lys
2105 2110 2115
Arg Lys Glu Glu
2120
<210> 8
<211> 2084
<212> PRT
<213> 口腔链球菌
<400> 8
Asp Gln Ala Arg Val Gly Ser Thr Asp Asn Leu Pro Ser Glu Leu Ala
1 5 10 15
Asp Leu Asp Lys Lys Ala Ser Asp Glu Gly His Asp Phe Asp Lys Glu
20 25 30
Ala Ala Ala Gln Asn Pro Gly Ser Ala Glu Thr Thr Glu Gly Pro Gln
35 40 45
Thr Glu Glu Glu Leu Leu Ala Gln Glu Lys Glu Lys Ser Glu Lys Pro
50 55 60
Ser Asn Leu Pro Lys Glu Leu Glu Asp Lys Leu Glu Lys Ala Glu Asp
65 70 75 80
Asn Gly Arg Glu Val Asp Lys Asp Gln Leu Ala Gln Asp Thr Gly Lys
85 90 95
Leu Val Pro Glu Asp Val Ala Lys Thr Thr Asn Gly Glu Leu Asn Tyr
100 105 110
Gly Ala Thr Val Lys Ile Lys Thr Pro Ser Gly Glu Gly Ser Gly Ile
115 120 125
Val Val Ala Lys Asp Leu Val Leu Thr Val Ser His Asn Phe Ile Lys
130 135 140
Asp Ser Gln Glu Gly Asn Ile Arg Lys Val Val Asp Asn Asp Gln Gly
145 150 155 160
Asp Gly Asp Ile Tyr Ser Ile Ser Tyr Pro Gly Leu Pro Asp Val Lys
165 170 175
Phe Ser Lys Lys Asp Ile Ile His Trp Asp Arg Glu Gly Tyr Leu Lys
180 185 190
Gly Phe Lys Asn Asp Leu Ala Leu Val Arg Leu Arg Thr Val Leu Glu
195 200 205
Asn Thr Pro Val Glu Val Thr Lys Lys Pro Val Val Lys Lys Ile Gly
210 215 220
Asp Lys Leu His Val Phe Gly Tyr Pro Glu Gly Lys Leu Asn Pro Ile
225 230 235 240
Val Asn Thr Thr Val Asp Phe Ala Glu Pro Tyr Gly Glu Gly Val Gln
245 250 255
Gly Ile Gly Tyr Gln Gly Gly Lys Pro Gly Ala Ser Gly Gly Gly Ile
260 265 270
Phe Asp Thr Glu Gly Lys Leu Val Gly Val His Gln Asn Gly Val Val
275 280 285
Gly Lys Arg Ser Gly Gly Ile Leu Phe Ser Pro Ala Gln Leu Lys Trp
290 295 300
Ile Gln Asp His Met Gln Gly Ile Ser Ser Val Lys Pro Ala Asp Leu
305 310 315 320
Glu Glu Lys Glu Lys Pro Ala Glu Glu Lys Pro Lys Glu Asp Lys Pro
325 330 335
Ala Ala Ala Lys Pro Glu Thr Pro Lys Ala Val Thr Pro Glu Trp Gln
340 345 350
Thr Val Ala Asn Lys Glu Gln Gln Gly Thr Val Thr Ile Arg Glu Glu
355 360 365
Lys Gly Val Arg Tyr Asn Gln Leu Ser Ser Thr Ala Gln Asn Asp Asn
370 375 380
Asp Gly Lys Pro Ala Leu Phe Glu Lys Gln Gly Leu Thr Val Asp Ala
385 390 395 400
Asn Gly Asn Ala Thr Val Asp Leu Thr Phe Lys Asp Asp Ser Glu Lys
405 410 415
Gly Lys Ser Arg Phe Gly Val Phe Leu Lys Phe Lys Asp Thr Lys Asn
420 425 430
Asn Val Phe Val Gly Tyr Asp Gln Gly Gly Trp Phe Trp Glu Tyr Lys
435 440 445
Thr Pro Gly Asn Ser Thr Trp Tyr Lys Gly Asn Arg Val Ala Ala Pro
450 455 460
Glu Pro Gly Ser Val Asn Arg Leu Ser Ile Thr Leu Lys Ser Asp Gly
465 470 475 480
Gln Leu Asn Ala Ser Asn Asn Asp Val Asn Leu Phe Asp Thr Val Thr
485 490 495
Leu Pro Gly Ala Val Asn Glu Asn Leu Lys Asn Glu Lys Lys Ile Leu
500 505 510
Leu Lys Ala Gly Thr Tyr Ser Asn Asp Arg Thr Val Val Ser Val Lys
515 520 525
Thr Asp Asn Gln Glu Gly Val Lys Ala Asp Asp Thr Pro Ala Gln Lys
530 535 540
Glu Thr Gly Pro Ala Val Asp Asp Ser Lys Val Thr Tyr Asp Thr Ile
545 550 555 560
Gln Ser Lys Val Leu Lys Ala Val Ile Asp Gln Ala Phe Pro Arg Val
565 570 575
Lys Glu Tyr Thr Leu Asn Gly His Thr Leu Pro Gly Gln Val Gln Gln
580 585 590
Phe Asn Gln Val Phe Ile Asn Asn His Arg Ile Thr Pro Glu Val Thr
595 600 605
Tyr Lys Lys Ile Asn Glu Thr Thr Ala Glu Tyr Leu Met Lys Leu Arg
610 615 620
Asp Asp Ala His Leu Ile Asn Ala Glu Met Thr Val Arg Leu Gln Val
625 630 635 640
Val Asp Asn Gln Leu His Phe Asp Val Thr Lys Ile Val Asn His Asn
645 650 655
Gln Val Thr Pro Gly Gln Lys Ile Asp Asp Glu Arg Lys Leu Leu Ser
660 665 670
Thr Ile Ser Phe Leu Gly Asn Ala Leu Val Ser Val Ser Ser Asp Gln
675 680 685
Ala Gly Ala Lys Phe Asp Gly Ala Thr Met Ser Asn Asn Thr His Val
690 695 700
Ser Gly Asp Asp His Ile Asp Val Thr Asn Pro Met Lys Asp Leu Ala
705 710 715 720
Lys Gly Tyr Met Tyr Gly Phe Val Ser Thr Asp Lys Leu Ala Ala Gly
725 730 735
Val Trp Ser Asn Ser Gln Asn Ser Tyr Gly Gly Gly Ser Asn Asp Trp
740 745 750
Thr Arg Leu Thr Ala Tyr Lys Glu Thr Val Gly Asn Ala Asn Tyr Val
755 760 765
Gly Ile His Ser Ser Glu Trp Gln Trp Glu Lys Ala Tyr Lys Gly Ile
770 775 780
Val Phe Pro Glu Tyr Thr Lys Glu Leu Pro Ser Ala Lys Val Val Ile
785 790 795 800
Thr Glu Asp Ala Asn Ala Asp Asn Lys Val Asp Trp Gln Asp Gly Ala
805 810 815
Ile Ala Tyr Arg Ser Ile Met Asn Asn Pro Gln Gly Trp Glu Lys Val
820 825 830
Lys Asp Ile Thr Ala Tyr Arg Ile Ala Met Asn Phe Gly Ser Gln Ala
835 840 845
Gln Asn Pro Phe Leu Met Thr Leu Asp Gly Ile Lys Lys Ile Asn Leu
850 855 860
His Thr Asp Gly Leu Gly Gln Gly Val Leu Leu Lys Gly Tyr Gly Ser
865 870 875 880
Glu Gly His Asp Ser Gly His Leu Asn Tyr Ala Asp Ile Gly Lys Arg
885 890 895
Ile Gly Gly Val Glu Asp Phe Lys Thr Leu Ile Glu Lys Ala Lys Lys
900 905 910
Tyr Gly Ala His Leu Gly Ile His Val Asn Ala Ser Glu Thr Tyr Pro
915 920 925
Glu Ser Lys Tyr Phe Asn Glu Asn Ile Leu Arg Lys Asn Pro Asp Gly
930 935 940
Ser Tyr Ser Tyr Gly Trp Asn Trp Leu Asp Gln Gly Ile Asn Ile Asp
945 950 955 960
Ala Ala Tyr Asp Leu Ala His Gly Arg Leu Ala Arg Trp Glu Asp Leu
965 970 975
Lys Lys Lys Leu Gly Glu Gly Leu Asp Phe Ile Tyr Val Asp Val Trp
980 985 990
Gly Asn Gly Gln Ser Gly Asp Asn Gly Ala Trp Ala Thr His Val Leu
995 1000 1005
Ala Lys Glu Ile Asn Lys Gln Gly Trp Arg Phe Ala Ile Glu Trp
1010 1015 1020
Gly His Gly Gly Glu Tyr Asp Ser Thr Phe Gln His Trp Ala Ala
1025 1030 1035
Asp Leu Thr Tyr Gly Gly Tyr Thr Asn Lys Gly Ile Asn Ser Ala
1040 1045 1050
Ile Thr Arg Phe Ile Arg Asn His Gln Lys Asp Ser Trp Val Gly
1055 1060 1065
Asp Tyr Arg Ser Tyr Gly Gly Ala Ala Asn Tyr Pro Leu Leu Gly
1070 1075 1080
Gly Tyr Ser Met Lys Asp Phe Glu Gly Trp Gln Gly Arg Ser Asp
1085 1090 1095
Tyr Asn Gly Tyr Val Thr Asn Leu Phe Ala His Asp Val Met Thr
1100 1105 1110
Lys Tyr Phe Gln His Phe Thr Val Ser Lys Trp Glu Asn Gly Thr
1115 1120 1125
Pro Val Thr Met Thr Asp Asn Gly Ser Thr Tyr Lys Trp Thr Pro
1130 1135 1140
Glu Met Lys Val Glu Leu Val Asp Ala Ala Gly Asn Lys Val Val
1145 1150 1155
Val Thr Arg Lys Ser Asn Asp Val Asn Ser Pro Gln Tyr Arg Glu
1160 1165 1170
Arg Thr Val Thr Leu Asn Gly Arg Val Ile Gln Asp Gly Ser Ala
1175 1180 1185
Tyr Leu Thr Pro Trp Asn Trp Asp Ala Asn Gly Lys Lys Leu Pro
1190 1195 1200
Thr Glu Lys Glu Lys Met Tyr Tyr Phe Asn Thr Gln Ala Gly Ala
1205 1210 1215
Thr Thr Trp Thr Leu Pro Ser Asp Trp Ala Asn Ser Lys Val Tyr
1220 1225 1230
Leu Tyr Lys Leu Thr Asp Gln Gly Lys Thr Glu Glu Gln Glu Leu
1235 1240 1245
Thr Val Thr Asp Gly Lys Ile Thr Leu Asp Leu Leu Ala Asn Gln
1250 1255 1260
Pro Tyr Val Leu Tyr Arg Ser Lys Gln Thr Asn Pro Glu Met Ser
1265 1270 1275
Trp Ser Glu Gly Met His Ile Tyr Asp Gln Gly Phe Asn Ser Gly
1280 1285 1290
Thr Leu Lys His Trp Thr Ile Ser Gly Asp Ala Ser Lys Ala Glu
1295 1300 1305
Ile Val Lys Ser Gln Gly Ala Asn Glu Met Leu Arg Ile Gln Gly
1310 1315 1320
Asn Lys Ser Lys Val Ser Leu Thr Gln Lys Leu Thr Gly Leu Lys
1325 1330 1335
Pro Asn Thr Lys Tyr Ala Val Tyr Val Gly Val Asp Asn Arg Ser
1340 1345 1350
Asn Ala Lys Ala Ser Ile Thr Val Asn Thr Gly Glu Lys Glu Val
1355 1360 1365
Thr Thr Tyr Thr Asn Lys Ser Leu Ala Leu Asn Tyr Ile Lys Ala
1370 1375 1380
Tyr Ala His Asn Asn Arg Arg Glu Asn Ala Thr Val Asp Asp Thr
1385 1390 1395
Ser Tyr Phe Gln Asn Met Tyr Ala Phe Phe Thr Thr Gly Ser Asp
1400 1405 1410
Val Ser Asn Val Thr Leu Thr Leu Ser Arg Glu Ala Gly Asp Glu
1415 1420 1425
Ala Thr Tyr Phe Asp Glu Ile Arg Thr Phe Glu Asn Asn Ser Ser
1430 1435 1440
Met Tyr Gly Asp Lys His Asp Thr Gly Gln Gly Thr Phe Lys Gln
1445 1450 1455
Asp Phe Glu Asn Val Ala Gln Gly Ile Phe Pro Phe Val Val Gly
1460 1465 1470
Gly Val Glu Gly Val Glu Asp Asn Arg Thr His Leu Ser Glu Lys
1475 1480 1485
His Asp Pro Tyr Thr Gln Arg Gly Trp Asn Gly Lys Lys Val Asp
1490 1495 1500
Asp Val Ile Glu Gly Asn Trp Ser Leu Lys Thr Asn Gly Leu Val
1505 1510 1515
Ser Arg Arg Asn Leu Val Tyr Gln Thr Ile Pro Gln Asn Phe Arg
1520 1525 1530
Phe Glu Ala Gly Lys Thr Tyr Arg Val Thr Phe Glu Tyr Glu Ala
1535 1540 1545
Gly Ser Asp Asn Thr Tyr Ala Phe Val Val Gly Lys Gly Glu Phe
1550 1555 1560
Gln Ser Gly Arg Arg Gly Thr Gln Ala Ser Asn Leu Glu Met His
1565 1570 1575
Glu Leu Pro Asn Thr Trp Thr Asp Ser Lys Lys Ala Lys Lys Val
1580 1585 1590
Thr Phe Leu Val Thr Gly Ala Glu Thr Gly Asp Thr Trp Val Gly
1595 1600 1605
Ile Tyr Ser Thr Gly Asn Ala Ser Asn Thr Arg Gly Asp Ala Gly
1610 1615 1620
Gly Asn Ala Asn Phe Arg Gly Tyr Asn Asp Phe Met Met Asp Asn
1625 1630 1635
Leu Gln Ile Glu Glu Ile Thr Leu Thr Gly Lys Met Leu Thr Glu
1640 1645 1650
Asn Ala Leu Lys Asn Tyr Leu Pro Thr Val Ala Met Thr Asn Tyr
1655 1660 1665
Thr Lys Glu Ser Met Asp Ala Leu Lys Glu Ala Val Phe Asn Leu
1670 1675 1680
Ser Gln Ala Asp Asp Asp Ile Ser Val Glu Glu Ala Arg Ala Glu
1685 1690 1695
Ile Ala Lys Ile Glu Ala Leu Lys Asn Ala Leu Val Gln Lys Lys
1700 1705 1710
Thr Ala Leu Val Ala Glu Asp Phe Glu Ser Leu Asp Ala Pro Ala
1715 1720 1725
Gln Pro Gly Glu Gly Leu Glu Asn Ala Phe Asp Gly Asn Val Ser
1730 1735 1740
Ser Leu Trp His Thr Ser Trp Asn Gly Gly Asp Val Gly Lys Pro
1745 1750 1755
Ala Thr Met Val Leu Lys Glu Pro Thr Glu Ile Thr Gly Leu Arg
1760 1765 1770
Tyr Val Pro Arg Ala Ser Asp Ser Asn Gly Asn Leu Arg Asp Val
1775 1780 1785
Lys Leu Val Val Thr Asp Glu Ser Gly Lys Glu His Thr Phe Asn
1790 1795 1800
Val Thr Asp Trp Pro Asn Asn Asn Lys Pro Lys Asp Ile Asp Phe
1805 1810 1815
Gly Lys Thr Ile Lys Ala Lys Lys Ile Val Leu Thr Gly Thr Lys
1820 1825 1830
Thr Tyr Gly Asp Gly Gly Asp Lys Tyr Gln Ser Ala Ala Glu Leu
1835 1840 1845
Ile Phe Thr Arg Pro Gln Val Ala Glu Thr Pro Leu Asp Leu Ser
1850 1855 1860
Gly Tyr Glu Ala Ala Leu Ala Lys Ala Gln Lys Leu Thr Asp Lys
1865 1870 1875
Asp Asn Gln Glu Glu Val Ala Ser Val Gln Ala Ser Met Lys Tyr
1880 1885 1890
Ala Thr Asp Asn His Leu Leu Thr Glu Arg Met Val Ala Tyr Phe
1895 1900 1905
Ala Asp Tyr Leu Asn Gln Leu Lys Asp Ser Ala Thr Lys Pro Asp
1910 1915 1920
Ala Pro Thr Ser Ser Lys Gly Glu Glu Gln Pro Pro Val Leu Asp
1925 1930 1935
Val Pro Glu Phe Lys Gly Gly Val Asn Ala Thr Glu Ala Ala Val
1940 1945 1950
His Glu Val Pro Glu Phe Lys Gly Gly Val Asn Ala Val Gln Ala
1955 1960 1965
Leu Val His Glu Leu Pro Glu Tyr Lys Gly Gly Ala Asn Ala Val
1970 1975 1980
Leu Ala Ala Ala Asn Glu Val Pro Glu Tyr Lys Gly Gly Ala Asn
1985 1990 1995
Ala Val Glu Ala Leu Val Asn Glu Lys Pro Ala Tyr Thr Gly Val
2000 2005 2010
Leu Ala Thr Ala Gly Asp Gln Ala Ala Pro Thr Val Glu Lys Pro
2015 2020 2025
Glu Tyr Pro Leu Thr Pro Ser Pro Val Ala Asp Thr Lys Thr Pro
2030 2035 2040
Gly Ala Lys Asp Glu Glu Lys Leu Pro Ala Thr Gly Glu His Ser
2045 2050 2055
Ser Glu Val Ala Leu Phe Leu Ala Ser Val Ser Ile Ala Leu Ser
2060 2065 2070
Ala Ala Val Leu Ala Thr Lys Arg Lys Glu Glu
2075 2080
<210> 9
<211> 2053
<212> PRT
<213> 口腔链球菌
<400> 9
Asp Gln Ala Arg Val Gly Ser Thr Asp Asn Leu Pro Ser Glu Leu Ala
1 5 10 15
Asp Leu Asp Lys Lys Ala Ser Asp Glu Gly His Asp Phe Asp Lys Glu
20 25 30
Ala Ala Ala Gln Asn Pro Gly Ser Ala Glu Thr Thr Glu Gly Pro Gln
35 40 45
Thr Glu Glu Glu Leu Leu Ala Gln Glu Lys Glu Lys Ser Glu Lys Pro
50 55 60
Ser Asn Leu Pro Lys Glu Leu Glu Asp Lys Leu Glu Lys Ala Glu Asp
65 70 75 80
Asn Gly Arg Glu Val Asp Lys Asp Gln Leu Ala Gln Asp Thr Gly Lys
85 90 95
Leu Val Pro Glu Asp Val Ala Lys Thr Thr Asn Gly Glu Leu Asn Tyr
100 105 110
Gly Ala Thr Val Lys Ile Lys Thr Pro Ser Gly Glu Gly Ser Gly Ile
115 120 125
Val Val Ala Lys Asp Leu Val Leu Thr Val Ser His Asn Phe Ile Lys
130 135 140
Asp Ser Gln Glu Gly Asn Ile Arg Lys Val Val Asp Asn Asp Gln Gly
145 150 155 160
Asp Gly Asp Ile Tyr Ser Ile Ser Tyr Pro Gly Leu Pro Asp Val Lys
165 170 175
Phe Ser Lys Lys Asp Ile Ile His Trp Asp Arg Glu Gly Tyr Leu Lys
180 185 190
Gly Phe Lys Asn Asp Leu Ala Leu Val Arg Leu Arg Thr Val Leu Glu
195 200 205
Asn Thr Pro Val Glu Val Thr Lys Lys Pro Val Val Lys Lys Ile Gly
210 215 220
Asp Lys Leu His Val Phe Gly Tyr Pro Glu Gly Lys Leu Asn Pro Ile
225 230 235 240
Val Asn Thr Thr Val Asp Phe Ala Glu Pro Tyr Gly Glu Gly Val Gln
245 250 255
Gly Ile Gly Tyr Gln Gly Gly Lys Pro Gly Ala Ser Gly Gly Gly Ile
260 265 270
Phe Asp Thr Glu Gly Lys Leu Val Gly Val His Gln Asn Gly Val Val
275 280 285
Gly Lys Arg Ser Gly Gly Ile Leu Phe Ser Pro Ala Gln Leu Lys Trp
290 295 300
Ile Gln Asp His Met Gln Gly Ile Ser Ser Val Lys Pro Ala Asp Leu
305 310 315 320
Glu Glu Lys Glu Lys Pro Ala Glu Glu Lys Pro Lys Glu Asp Lys Pro
325 330 335
Ala Ala Ala Lys Pro Glu Thr Pro Lys Ala Val Thr Pro Glu Trp Gln
340 345 350
Thr Val Ala Asn Lys Glu Gln Gln Gly Thr Val Thr Ile Arg Glu Glu
355 360 365
Lys Gly Val Arg Tyr Asn Gln Leu Ser Ser Thr Ala Gln Asn Asp Asn
370 375 380
Asp Gly Lys Pro Ala Leu Phe Glu Lys Gln Gly Leu Thr Val Asp Ala
385 390 395 400
Asn Gly Asn Ala Thr Val Asp Leu Thr Phe Lys Asp Asp Ser Glu Lys
405 410 415
Gly Lys Ser Arg Phe Gly Val Phe Leu Lys Phe Lys Asp Thr Lys Asn
420 425 430
Asn Val Phe Val Gly Tyr Asp Gln Gly Gly Trp Phe Trp Glu Tyr Lys
435 440 445
Thr Pro Gly Asn Ser Thr Trp Tyr Lys Gly Asn Arg Val Ala Ala Pro
450 455 460
Glu Pro Gly Ser Val Asn Arg Leu Ser Ile Thr Leu Lys Ser Asp Gly
465 470 475 480
Gln Leu Asn Ala Ser Asn Asn Asp Val Asn Leu Phe Asp Thr Val Thr
485 490 495
Leu Pro Gly Ala Val Asn Glu Asn Leu Lys Asn Glu Lys Lys Ile Leu
500 505 510
Leu Lys Ala Gly Thr Tyr Ser Asn Asp Arg Thr Val Val Ser Val Lys
515 520 525
Thr Asp Asn Gln Glu Gly Val Lys Ala Asp Asp Thr Pro Ala Gln Lys
530 535 540
Glu Thr Gly Pro Ala Val Asp Asp Ser Lys Val Thr Tyr Asp Thr Ile
545 550 555 560
Gln Ser Lys Val Leu Lys Ala Val Ile Asp Gln Ala Phe Pro Arg Val
565 570 575
Lys Glu Tyr Thr Leu Asn Gly His Thr Leu Pro Gly Gln Val Gln Gln
580 585 590
Phe Asn Gln Val Phe Ile Asn Asn His Arg Ile Thr Pro Glu Val Thr
595 600 605
Tyr Lys Lys Ile Asn Glu Thr Thr Ala Glu Tyr Leu Met Lys Leu Arg
610 615 620
Asp Asp Ala His Leu Ile Asn Ala Glu Met Thr Val Arg Leu Gln Val
625 630 635 640
Val Asp Asn Gln Leu His Phe Asp Val Thr Lys Ile Val Asn His Asn
645 650 655
Gln Val Thr Pro Gly Gln Lys Ile Asp Asp Glu Arg Lys Leu Leu Ser
660 665 670
Thr Ile Ser Phe Leu Gly Asn Ala Leu Val Ser Val Ser Ser Asp Gln
675 680 685
Ala Gly Ala Lys Phe Asp Gly Ala Thr Met Ser Asn Asn Thr His Val
690 695 700
Ser Gly Asp Asp His Ile Asp Val Thr Asn Pro Met Lys Asp Leu Ala
705 710 715 720
Lys Gly Tyr Met Tyr Gly Phe Val Ser Thr Asp Lys Leu Ala Ala Gly
725 730 735
Val Trp Ser Asn Ser Gln Asn Ser Tyr Gly Gly Gly Ser Asn Asp Trp
740 745 750
Thr Arg Leu Thr Ala Tyr Lys Glu Thr Val Gly Asn Ala Asn Tyr Val
755 760 765
Gly Ile His Ser Ser Glu Trp Gln Trp Glu Lys Ala Tyr Lys Gly Ile
770 775 780
Val Phe Pro Glu Tyr Thr Lys Glu Leu Pro Ser Ala Lys Val Val Ile
785 790 795 800
Thr Glu Asp Ala Asn Ala Asp Asn Lys Val Asp Trp Gln Asp Gly Ala
805 810 815
Ile Ala Tyr Arg Ser Ile Met Asn Asn Pro Gln Gly Trp Glu Lys Val
820 825 830
Lys Asp Ile Thr Ala Tyr Arg Ile Ala Met Asn Phe Gly Ser Gln Ala
835 840 845
Gln Asn Pro Phe Leu Met Thr Leu Asp Gly Ile Lys Lys Ile Asn Leu
850 855 860
His Thr Asp Gly Leu Gly Gln Gly Val Leu Leu Lys Gly Tyr Gly Ser
865 870 875 880
Glu Gly His Asp Ser Gly His Leu Asn Tyr Ala Asp Ile Gly Lys Arg
885 890 895
Ile Gly Gly Val Glu Asp Phe Lys Thr Leu Ile Glu Lys Ala Lys Lys
900 905 910
Tyr Gly Ala His Leu Gly Ile His Val Asn Ala Ser Glu Thr Tyr Pro
915 920 925
Glu Ser Lys Tyr Phe Asn Glu Asn Ile Leu Arg Lys Asn Pro Asp Gly
930 935 940
Ser Tyr Ser Tyr Gly Trp Asn Trp Leu Asp Gln Gly Ile Asn Ile Asp
945 950 955 960
Ala Ala Tyr Asp Leu Ala His Gly Arg Leu Ala Arg Trp Glu Asp Leu
965 970 975
Lys Lys Lys Leu Gly Glu Gly Leu Asp Phe Ile Tyr Val Asp Val Trp
980 985 990
Gly Asn Gly Gln Ser Gly Asp Asn Gly Ala Trp Ala Thr His Val Leu
995 1000 1005
Ala Lys Glu Ile Asn Lys Gln Gly Trp Arg Phe Ala Ile Glu Trp
1010 1015 1020
Gly His Gly Gly Glu Tyr Asp Ser Thr Phe Gln His Trp Ala Ala
1025 1030 1035
Asp Leu Thr Tyr Gly Gly Tyr Thr Asn Lys Gly Ile Asn Ser Ala
1040 1045 1050
Ile Thr Arg Phe Ile Arg Asn His Gln Lys Asp Ser Trp Val Gly
1055 1060 1065
Asp Tyr Arg Ser Tyr Gly Gly Ala Ala Asn Tyr Pro Leu Leu Gly
1070 1075 1080
Gly Tyr Ser Met Lys Asp Phe Glu Gly Trp Gln Gly Arg Ser Asp
1085 1090 1095
Tyr Asn Gly Tyr Val Thr Asn Leu Phe Ala His Asp Val Met Thr
1100 1105 1110
Lys Tyr Phe Gln His Phe Thr Val Ser Lys Trp Glu Asn Gly Thr
1115 1120 1125
Pro Val Thr Met Thr Asp Asn Gly Ser Thr Tyr Lys Trp Thr Pro
1130 1135 1140
Glu Met Lys Val Glu Leu Val Asp Ala Ala Gly Asn Lys Val Val
1145 1150 1155
Val Thr Arg Lys Ser Asn Asp Val Asn Ser Pro Gln Tyr Arg Glu
1160 1165 1170
Arg Thr Val Thr Leu Asn Gly Arg Val Ile Gln Asp Gly Ser Ala
1175 1180 1185
Tyr Leu Thr Pro Trp Asn Trp Asp Ala Asn Gly Lys Lys Leu Pro
1190 1195 1200
Thr Glu Lys Glu Lys Met Tyr Tyr Phe Asn Thr Gln Ala Gly Ala
1205 1210 1215
Thr Thr Trp Thr Leu Pro Ser Asp Trp Ala Asn Ser Lys Val Tyr
1220 1225 1230
Leu Tyr Lys Leu Thr Asp Gln Gly Lys Thr Glu Glu Gln Glu Leu
1235 1240 1245
Thr Val Thr Asp Gly Lys Ile Thr Leu Asp Leu Leu Ala Asn Gln
1250 1255 1260
Pro Tyr Val Leu Tyr Arg Ser Lys Gln Thr Asn Pro Glu Met Ser
1265 1270 1275
Trp Ser Glu Gly Met His Ile Tyr Asp Gln Gly Phe Asn Ser Gly
1280 1285 1290
Thr Leu Lys His Trp Thr Ile Ser Gly Asp Ala Ser Lys Ala Glu
1295 1300 1305
Ile Val Lys Ser Gln Gly Ala Asn Glu Met Leu Arg Ile Gln Gly
1310 1315 1320
Asn Lys Ser Lys Val Ser Leu Thr Gln Lys Leu Thr Gly Leu Lys
1325 1330 1335
Pro Asn Thr Lys Tyr Ala Val Tyr Val Gly Val Asp Asn Arg Ser
1340 1345 1350
Asn Ala Lys Ala Ser Ile Thr Val Asn Thr Gly Glu Lys Glu Val
1355 1360 1365
Thr Thr Tyr Thr Asn Lys Ser Leu Ala Leu Asn Tyr Ile Lys Ala
1370 1375 1380
Tyr Ala His Asn Asn Arg Arg Glu Asn Ala Thr Val Asp Asp Thr
1385 1390 1395
Ser Tyr Phe Gln Asn Met Tyr Ala Phe Phe Thr Thr Gly Ser Asp
1400 1405 1410
Val Ser Asn Val Thr Leu Thr Leu Ser Arg Glu Ala Gly Asp Glu
1415 1420 1425
Ala Thr Tyr Phe Asp Glu Ile Arg Thr Phe Glu Asn Asn Ser Ser
1430 1435 1440
Met Tyr Gly Asp Lys His Asp Thr Gly Gln Gly Thr Phe Lys Gln
1445 1450 1455
Asp Phe Glu Asn Val Ala Gln Gly Ile Phe Pro Phe Val Val Gly
1460 1465 1470
Gly Val Glu Gly Val Glu Asp Asn Arg Thr His Leu Ser Glu Lys
1475 1480 1485
His Asp Pro Tyr Thr Gln Arg Gly Trp Asn Gly Lys Lys Val Asp
1490 1495 1500
Asp Val Ile Glu Gly Asn Trp Ser Leu Lys Thr Asn Gly Leu Val
1505 1510 1515
Ser Arg Arg Asn Leu Val Tyr Gln Thr Ile Pro Gln Asn Phe Arg
1520 1525 1530
Phe Glu Ala Gly Lys Thr Tyr Arg Val Thr Phe Glu Tyr Glu Ala
1535 1540 1545
Gly Ser Asp Asn Thr Tyr Ala Phe Val Val Gly Lys Gly Glu Phe
1550 1555 1560
Gln Ser Gly Arg Arg Gly Thr Gln Ala Ser Asn Leu Glu Met His
1565 1570 1575
Glu Leu Pro Asn Thr Trp Thr Asp Ser Lys Lys Ala Lys Lys Val
1580 1585 1590
Thr Phe Leu Val Thr Gly Ala Glu Thr Gly Asp Thr Trp Val Gly
1595 1600 1605
Ile Tyr Ser Thr Gly Asn Ala Ser Asn Thr Arg Gly Asp Ala Gly
1610 1615 1620
Gly Asn Ala Asn Phe Arg Gly Tyr Asn Asp Phe Met Met Asp Asn
1625 1630 1635
Leu Gln Ile Glu Glu Ile Thr Leu Thr Gly Lys Met Leu Thr Glu
1640 1645 1650
Asn Ala Leu Lys Asn Tyr Leu Pro Thr Val Ala Met Thr Asn Tyr
1655 1660 1665
Thr Lys Glu Ser Met Asp Ala Leu Lys Glu Ala Val Phe Asn Leu
1670 1675 1680
Ser Gln Ala Asp Asp Asp Ile Ser Val Glu Glu Ala Arg Ala Glu
1685 1690 1695
Ile Ala Lys Ile Glu Ala Leu Lys Asn Ala Leu Val Gln Lys Lys
1700 1705 1710
Thr Ala Leu Val Ala Glu Asp Phe Glu Ser Leu Asp Ala Pro Ala
1715 1720 1725
Gln Pro Gly Glu Gly Leu Glu Asn Ala Phe Asp Gly Asn Val Ser
1730 1735 1740
Ser Leu Trp His Thr Ser Trp Asn Gly Gly Asp Val Gly Lys Pro
1745 1750 1755
Ala Thr Met Val Leu Lys Glu Pro Thr Glu Ile Thr Gly Leu Arg
1760 1765 1770
Tyr Val Pro Arg Ala Ser Asp Ser Asn Gly Asn Leu Arg Asp Val
1775 1780 1785
Lys Leu Val Val Thr Asp Glu Ser Gly Lys Glu His Thr Phe Asn
1790 1795 1800
Val Thr Asp Trp Pro Asn Asn Asn Lys Pro Lys Asp Ile Asp Phe
1805 1810 1815
Gly Lys Thr Ile Lys Ala Lys Lys Ile Val Leu Thr Gly Thr Lys
1820 1825 1830
Thr Tyr Gly Asp Gly Gly Asp Lys Tyr Gln Ser Ala Ala Glu Leu
1835 1840 1845
Ile Phe Thr Arg Pro Gln Val Ala Glu Thr Pro Leu Asp Leu Ser
1850 1855 1860
Gly Tyr Glu Ala Ala Leu Ala Lys Ala Gln Lys Leu Thr Asp Lys
1865 1870 1875
Asp Asn Gln Glu Glu Val Ala Ser Val Gln Ala Ser Met Lys Tyr
1880 1885 1890
Ala Thr Asp Asn His Leu Leu Thr Glu Arg Met Val Ala Tyr Phe
1895 1900 1905
Ala Asp Tyr Leu Asn Gln Leu Lys Asp Ser Ala Thr Lys Pro Asp
1910 1915 1920
Ala Pro Thr Ser Ser Lys Gly Glu Glu Gln Pro Pro Val Leu Asp
1925 1930 1935
Val Pro Glu Phe Lys Gly Gly Val Asn Ala Thr Glu Ala Ala Val
1940 1945 1950
His Glu Val Pro Glu Phe Lys Gly Gly Val Asn Ala Val Gln Ala
1955 1960 1965
Leu Val His Glu Leu Pro Glu Tyr Lys Gly Gly Ala Asn Ala Val
1970 1975 1980
Leu Ala Ala Ala Asn Glu Val Pro Glu Tyr Lys Gly Gly Ala Asn
1985 1990 1995
Ala Val Glu Ala Leu Val Asn Glu Lys Pro Ala Tyr Thr Gly Val
2000 2005 2010
Leu Ala Thr Ala Gly Asp Gln Ala Ala Pro Thr Val Glu Lys Pro
2015 2020 2025
Glu Tyr Pro Leu Thr Pro Ser Pro Val Ala Asp Thr Lys Thr Pro
2030 2035 2040
Gly Ala Lys Asp Glu Glu Lys Leu Pro Ala
2045 2050
<210> 10
<211> 2065
<212> PRT
<213> 人工序列
<220>
<223> 来自口腔链球菌的O-糖苷酶,具有另外的N末端Met和C末端GSGLE-His6标签并去除信号序列和LPXTG细胞壁锚定基序的C末端元件
<400> 10
Met Asp Gln Ala Arg Val Gly Ser Thr Asp Asn Leu Pro Ser Glu Leu
1 5 10 15
Ala Asp Leu Asp Lys Lys Ala Ser Asp Glu Gly His Asp Phe Asp Lys
20 25 30
Glu Ala Ala Ala Gln Asn Pro Gly Ser Ala Glu Thr Thr Glu Gly Pro
35 40 45
Gln Thr Glu Glu Glu Leu Leu Ala Gln Glu Lys Glu Lys Ser Glu Lys
50 55 60
Pro Ser Asn Leu Pro Lys Glu Leu Glu Asp Lys Leu Glu Lys Ala Glu
65 70 75 80
Asp Asn Gly Arg Glu Val Asp Lys Asp Gln Leu Ala Gln Asp Thr Gly
85 90 95
Lys Leu Val Pro Glu Asp Val Ala Lys Thr Thr Asn Gly Glu Leu Asn
100 105 110
Tyr Gly Ala Thr Val Lys Ile Lys Thr Pro Ser Gly Glu Gly Ser Gly
115 120 125
Ile Val Val Ala Lys Asp Leu Val Leu Thr Val Ser His Asn Phe Ile
130 135 140
Lys Asp Ser Gln Glu Gly Asn Ile Arg Lys Val Val Asp Asn Asp Gln
145 150 155 160
Gly Asp Gly Asp Ile Tyr Ser Ile Ser Tyr Pro Gly Leu Pro Asp Val
165 170 175
Lys Phe Ser Lys Lys Asp Ile Ile His Trp Asp Arg Glu Gly Tyr Leu
180 185 190
Lys Gly Phe Lys Asn Asp Leu Ala Leu Val Arg Leu Arg Thr Val Leu
195 200 205
Glu Asn Thr Pro Val Glu Val Thr Lys Lys Pro Val Val Lys Lys Ile
210 215 220
Gly Asp Lys Leu His Val Phe Gly Tyr Pro Glu Gly Lys Leu Asn Pro
225 230 235 240
Ile Val Asn Thr Thr Val Asp Phe Ala Glu Pro Tyr Gly Glu Gly Val
245 250 255
Gln Gly Ile Gly Tyr Gln Gly Gly Lys Pro Gly Ala Ser Gly Gly Gly
260 265 270
Ile Phe Asp Thr Glu Gly Lys Leu Val Gly Val His Gln Asn Gly Val
275 280 285
Val Gly Lys Arg Ser Gly Gly Ile Leu Phe Ser Pro Ala Gln Leu Lys
290 295 300
Trp Ile Gln Asp His Met Gln Gly Ile Ser Ser Val Lys Pro Ala Asp
305 310 315 320
Leu Glu Glu Lys Glu Lys Pro Ala Glu Glu Lys Pro Lys Glu Asp Lys
325 330 335
Pro Ala Ala Ala Lys Pro Glu Thr Pro Lys Ala Val Thr Pro Glu Trp
340 345 350
Gln Thr Val Ala Asn Lys Glu Gln Gln Gly Thr Val Thr Ile Arg Glu
355 360 365
Glu Lys Gly Val Arg Tyr Asn Gln Leu Ser Ser Thr Ala Gln Asn Asp
370 375 380
Asn Asp Gly Lys Pro Ala Leu Phe Glu Lys Gln Gly Leu Thr Val Asp
385 390 395 400
Ala Asn Gly Asn Ala Thr Val Asp Leu Thr Phe Lys Asp Asp Ser Glu
405 410 415
Lys Gly Lys Ser Arg Phe Gly Val Phe Leu Lys Phe Lys Asp Thr Lys
420 425 430
Asn Asn Val Phe Val Gly Tyr Asp Gln Gly Gly Trp Phe Trp Glu Tyr
435 440 445
Lys Thr Pro Gly Asn Ser Thr Trp Tyr Lys Gly Asn Arg Val Ala Ala
450 455 460
Pro Glu Pro Gly Ser Val Asn Arg Leu Ser Ile Thr Leu Lys Ser Asp
465 470 475 480
Gly Gln Leu Asn Ala Ser Asn Asn Asp Val Asn Leu Phe Asp Thr Val
485 490 495
Thr Leu Pro Gly Ala Val Asn Glu Asn Leu Lys Asn Glu Lys Lys Ile
500 505 510
Leu Leu Lys Ala Gly Thr Tyr Ser Asn Asp Arg Thr Val Val Ser Val
515 520 525
Lys Thr Asp Asn Gln Glu Gly Val Lys Ala Asp Asp Thr Pro Ala Gln
530 535 540
Lys Glu Thr Gly Pro Ala Val Asp Asp Ser Lys Val Thr Tyr Asp Thr
545 550 555 560
Ile Gln Ser Lys Val Leu Lys Ala Val Ile Asp Gln Ala Phe Pro Arg
565 570 575
Val Lys Glu Tyr Thr Leu Asn Gly His Thr Leu Pro Gly Gln Val Gln
580 585 590
Gln Phe Asn Gln Val Phe Ile Asn Asn His Arg Ile Thr Pro Glu Val
595 600 605
Thr Tyr Lys Lys Ile Asn Glu Thr Thr Ala Glu Tyr Leu Met Lys Leu
610 615 620
Arg Asp Asp Ala His Leu Ile Asn Ala Glu Met Thr Val Arg Leu Gln
625 630 635 640
Val Val Asp Asn Gln Leu His Phe Asp Val Thr Lys Ile Val Asn His
645 650 655
Asn Gln Val Thr Pro Gly Gln Lys Ile Asp Asp Glu Arg Lys Leu Leu
660 665 670
Ser Thr Ile Ser Phe Leu Gly Asn Ala Leu Val Ser Val Ser Ser Asp
675 680 685
Gln Ala Gly Ala Lys Phe Asp Gly Ala Thr Met Ser Asn Asn Thr His
690 695 700
Val Ser Gly Asp Asp His Ile Asp Val Thr Asn Pro Met Lys Asp Leu
705 710 715 720
Ala Lys Gly Tyr Met Tyr Gly Phe Val Ser Thr Asp Lys Leu Ala Ala
725 730 735
Gly Val Trp Ser Asn Ser Gln Asn Ser Tyr Gly Gly Gly Ser Asn Asp
740 745 750
Trp Thr Arg Leu Thr Ala Tyr Lys Glu Thr Val Gly Asn Ala Asn Tyr
755 760 765
Val Gly Ile His Ser Ser Glu Trp Gln Trp Glu Lys Ala Tyr Lys Gly
770 775 780
Ile Val Phe Pro Glu Tyr Thr Lys Glu Leu Pro Ser Ala Lys Val Val
785 790 795 800
Ile Thr Glu Asp Ala Asn Ala Asp Asn Lys Val Asp Trp Gln Asp Gly
805 810 815
Ala Ile Ala Tyr Arg Ser Ile Met Asn Asn Pro Gln Gly Trp Glu Lys
820 825 830
Val Lys Asp Ile Thr Ala Tyr Arg Ile Ala Met Asn Phe Gly Ser Gln
835 840 845
Ala Gln Asn Pro Phe Leu Met Thr Leu Asp Gly Ile Lys Lys Ile Asn
850 855 860
Leu His Thr Asp Gly Leu Gly Gln Gly Val Leu Leu Lys Gly Tyr Gly
865 870 875 880
Ser Glu Gly His Asp Ser Gly His Leu Asn Tyr Ala Asp Ile Gly Lys
885 890 895
Arg Ile Gly Gly Val Glu Asp Phe Lys Thr Leu Ile Glu Lys Ala Lys
900 905 910
Lys Tyr Gly Ala His Leu Gly Ile His Val Asn Ala Ser Glu Thr Tyr
915 920 925
Pro Glu Ser Lys Tyr Phe Asn Glu Asn Ile Leu Arg Lys Asn Pro Asp
930 935 940
Gly Ser Tyr Ser Tyr Gly Trp Asn Trp Leu Asp Gln Gly Ile Asn Ile
945 950 955 960
Asp Ala Ala Tyr Asp Leu Ala His Gly Arg Leu Ala Arg Trp Glu Asp
965 970 975
Leu Lys Lys Lys Leu Gly Glu Gly Leu Asp Phe Ile Tyr Val Asp Val
980 985 990
Trp Gly Asn Gly Gln Ser Gly Asp Asn Gly Ala Trp Ala Thr His Val
995 1000 1005
Leu Ala Lys Glu Ile Asn Lys Gln Gly Trp Arg Phe Ala Ile Glu
1010 1015 1020
Trp Gly His Gly Gly Glu Tyr Asp Ser Thr Phe Gln His Trp Ala
1025 1030 1035
Ala Asp Leu Thr Tyr Gly Gly Tyr Thr Asn Lys Gly Ile Asn Ser
1040 1045 1050
Ala Ile Thr Arg Phe Ile Arg Asn His Gln Lys Asp Ser Trp Val
1055 1060 1065
Gly Asp Tyr Arg Ser Tyr Gly Gly Ala Ala Asn Tyr Pro Leu Leu
1070 1075 1080
Gly Gly Tyr Ser Met Lys Asp Phe Glu Gly Trp Gln Gly Arg Ser
1085 1090 1095
Asp Tyr Asn Gly Tyr Val Thr Asn Leu Phe Ala His Asp Val Met
1100 1105 1110
Thr Lys Tyr Phe Gln His Phe Thr Val Ser Lys Trp Glu Asn Gly
1115 1120 1125
Thr Pro Val Thr Met Thr Asp Asn Gly Ser Thr Tyr Lys Trp Thr
1130 1135 1140
Pro Glu Met Lys Val Glu Leu Val Asp Ala Ala Gly Asn Lys Val
1145 1150 1155
Val Val Thr Arg Lys Ser Asn Asp Val Asn Ser Pro Gln Tyr Arg
1160 1165 1170
Glu Arg Thr Val Thr Leu Asn Gly Arg Val Ile Gln Asp Gly Ser
1175 1180 1185
Ala Tyr Leu Thr Pro Trp Asn Trp Asp Ala Asn Gly Lys Lys Leu
1190 1195 1200
Pro Thr Glu Lys Glu Lys Met Tyr Tyr Phe Asn Thr Gln Ala Gly
1205 1210 1215
Ala Thr Thr Trp Thr Leu Pro Ser Asp Trp Ala Asn Ser Lys Val
1220 1225 1230
Tyr Leu Tyr Lys Leu Thr Asp Gln Gly Lys Thr Glu Glu Gln Glu
1235 1240 1245
Leu Thr Val Thr Asp Gly Lys Ile Thr Leu Asp Leu Leu Ala Asn
1250 1255 1260
Gln Pro Tyr Val Leu Tyr Arg Ser Lys Gln Thr Asn Pro Glu Met
1265 1270 1275
Ser Trp Ser Glu Gly Met His Ile Tyr Asp Gln Gly Phe Asn Ser
1280 1285 1290
Gly Thr Leu Lys His Trp Thr Ile Ser Gly Asp Ala Ser Lys Ala
1295 1300 1305
Glu Ile Val Lys Ser Gln Gly Ala Asn Glu Met Leu Arg Ile Gln
1310 1315 1320
Gly Asn Lys Ser Lys Val Ser Leu Thr Gln Lys Leu Thr Gly Leu
1325 1330 1335
Lys Pro Asn Thr Lys Tyr Ala Val Tyr Val Gly Val Asp Asn Arg
1340 1345 1350
Ser Asn Ala Lys Ala Ser Ile Thr Val Asn Thr Gly Glu Lys Glu
1355 1360 1365
Val Thr Thr Tyr Thr Asn Lys Ser Leu Ala Leu Asn Tyr Ile Lys
1370 1375 1380
Ala Tyr Ala His Asn Asn Arg Arg Glu Asn Ala Thr Val Asp Asp
1385 1390 1395
Thr Ser Tyr Phe Gln Asn Met Tyr Ala Phe Phe Thr Thr Gly Ser
1400 1405 1410
Asp Val Ser Asn Val Thr Leu Thr Leu Ser Arg Glu Ala Gly Asp
1415 1420 1425
Glu Ala Thr Tyr Phe Asp Glu Ile Arg Thr Phe Glu Asn Asn Ser
1430 1435 1440
Ser Met Tyr Gly Asp Lys His Asp Thr Gly Gln Gly Thr Phe Lys
1445 1450 1455
Gln Asp Phe Glu Asn Val Ala Gln Gly Ile Phe Pro Phe Val Val
1460 1465 1470
Gly Gly Val Glu Gly Val Glu Asp Asn Arg Thr His Leu Ser Glu
1475 1480 1485
Lys His Asp Pro Tyr Thr Gln Arg Gly Trp Asn Gly Lys Lys Val
1490 1495 1500
Asp Asp Val Ile Glu Gly Asn Trp Ser Leu Lys Thr Asn Gly Leu
1505 1510 1515
Val Ser Arg Arg Asn Leu Val Tyr Gln Thr Ile Pro Gln Asn Phe
1520 1525 1530
Arg Phe Glu Ala Gly Lys Thr Tyr Arg Val Thr Phe Glu Tyr Glu
1535 1540 1545
Ala Gly Ser Asp Asn Thr Tyr Ala Phe Val Val Gly Lys Gly Glu
1550 1555 1560
Phe Gln Ser Gly Arg Arg Gly Thr Gln Ala Ser Asn Leu Glu Met
1565 1570 1575
His Glu Leu Pro Asn Thr Trp Thr Asp Ser Lys Lys Ala Lys Lys
1580 1585 1590
Val Thr Phe Leu Val Thr Gly Ala Glu Thr Gly Asp Thr Trp Val
1595 1600 1605
Gly Ile Tyr Ser Thr Gly Asn Ala Ser Asn Thr Arg Gly Asp Ala
1610 1615 1620
Gly Gly Asn Ala Asn Phe Arg Gly Tyr Asn Asp Phe Met Met Asp
1625 1630 1635
Asn Leu Gln Ile Glu Glu Ile Thr Leu Thr Gly Lys Met Leu Thr
1640 1645 1650
Glu Asn Ala Leu Lys Asn Tyr Leu Pro Thr Val Ala Met Thr Asn
1655 1660 1665
Tyr Thr Lys Glu Ser Met Asp Ala Leu Lys Glu Ala Val Phe Asn
1670 1675 1680
Leu Ser Gln Ala Asp Asp Asp Ile Ser Val Glu Glu Ala Arg Ala
1685 1690 1695
Glu Ile Ala Lys Ile Glu Ala Leu Lys Asn Ala Leu Val Gln Lys
1700 1705 1710
Lys Thr Ala Leu Val Ala Glu Asp Phe Glu Ser Leu Asp Ala Pro
1715 1720 1725
Ala Gln Pro Gly Glu Gly Leu Glu Asn Ala Phe Asp Gly Asn Val
1730 1735 1740
Ser Ser Leu Trp His Thr Ser Trp Asn Gly Gly Asp Val Gly Lys
1745 1750 1755
Pro Ala Thr Met Val Leu Lys Glu Pro Thr Glu Ile Thr Gly Leu
1760 1765 1770
Arg Tyr Val Pro Arg Ala Ser Asp Ser Asn Gly Asn Leu Arg Asp
1775 1780 1785
Val Lys Leu Val Val Thr Asp Glu Ser Gly Lys Glu His Thr Phe
1790 1795 1800
Asn Val Thr Asp Trp Pro Asn Asn Asn Lys Pro Lys Asp Ile Asp
1805 1810 1815
Phe Gly Lys Thr Ile Lys Ala Lys Lys Ile Val Leu Thr Gly Thr
1820 1825 1830
Lys Thr Tyr Gly Asp Gly Gly Asp Lys Tyr Gln Ser Ala Ala Glu
1835 1840 1845
Leu Ile Phe Thr Arg Pro Gln Val Ala Glu Thr Pro Leu Asp Leu
1850 1855 1860
Ser Gly Tyr Glu Ala Ala Leu Ala Lys Ala Gln Lys Leu Thr Asp
1865 1870 1875
Lys Asp Asn Gln Glu Glu Val Ala Ser Val Gln Ala Ser Met Lys
1880 1885 1890
Tyr Ala Thr Asp Asn His Leu Leu Thr Glu Arg Met Val Ala Tyr
1895 1900 1905
Phe Ala Asp Tyr Leu Asn Gln Leu Lys Asp Ser Ala Thr Lys Pro
1910 1915 1920
Asp Ala Pro Thr Ser Ser Lys Gly Glu Glu Gln Pro Pro Val Leu
1925 1930 1935
Asp Val Pro Glu Phe Lys Gly Gly Val Asn Ala Thr Glu Ala Ala
1940 1945 1950
Val His Glu Val Pro Glu Phe Lys Gly Gly Val Asn Ala Val Gln
1955 1960 1965
Ala Leu Val His Glu Leu Pro Glu Tyr Lys Gly Gly Ala Asn Ala
1970 1975 1980
Val Leu Ala Ala Ala Asn Glu Val Pro Glu Tyr Lys Gly Gly Ala
1985 1990 1995
Asn Ala Val Glu Ala Leu Val Asn Glu Lys Pro Ala Tyr Thr Gly
2000 2005 2010
Val Leu Ala Thr Ala Gly Asp Gln Ala Ala Pro Thr Val Glu Lys
2015 2020 2025
Pro Glu Tyr Pro Leu Thr Pro Ser Pro Val Ala Asp Thr Lys Thr
2030 2035 2040
Pro Gly Ala Lys Asp Glu Glu Lys Leu Pro Ala Gly Ser Gly Leu
2045 2050 2055
Glu His His His His His His
2060 2065
<210> 11
<211> 361
<212> PRT
<213> 嗜粘蛋白艾克曼菌
<400> 11
Glu Val Thr Val Pro Asp Ala Leu Lys Asp Arg Ile Ala Leu Lys Lys
1 5 10 15
Thr Ala Arg Gln Leu Asn Ile Val Tyr Phe Leu Gly Ser Asp Thr Glu
20 25 30
Pro Val Pro Asp Tyr Glu Arg Arg Leu Ser Glu Leu Leu Leu Tyr Leu
35 40 45
Gln Gln Phe Tyr Gly Lys Glu Met Gln Arg His Gly Tyr Gly Ala Arg
50 55 60
Ser Phe Gly Leu Asp Ile Lys Ser Pro Gly Arg Val Asn Ile Ile Glu
65 70 75 80
Tyr Lys Ala Lys Asn Pro Ala Ala His Tyr Pro Tyr Glu Asn Gly Gly
85 90 95
Gly Trp Lys Ala Ala Gln Glu Leu Asp Glu Phe Phe Lys Ala His Pro
100 105 110
Asp Arg Lys Lys Ser Gln His Thr Leu Ile Ile Met Pro Thr Trp Asn
115 120 125
Asp Glu Lys Asn Gly Pro Asp Asn Pro Gly Gly Val Pro Phe Tyr Gly
130 135 140
Met Gly Arg Asn Cys Phe Ala Leu Asp Tyr Pro Ala Phe Asp Ile Lys
145 150 155 160
His Leu Gly Gln Lys Thr Arg Glu Gly Arg Leu Leu Thr Lys Trp Tyr
165 170 175
Gly Gly Met Ala His Glu Leu Gly His Gly Leu Asn Leu Pro His Asn
180 185 190
His Gln Thr Ala Ser Asp Gly Lys Lys Tyr Gly Thr Ala Leu Met Gly
195 200 205
Ser Gly Asn Tyr Thr Phe Gly Thr Ser Pro Thr Phe Leu Thr Pro Ala
210 215 220
Ser Cys Ala Leu Leu Asp Ala Cys Glu Val Phe Ser Val Thr Pro Ser
225 230 235 240
Gln Gln Phe Tyr Glu Gly Lys Pro Glu Val Glu Val Gly Asp Val Ala
245 250 255
Ile Ser Phe Lys Gly Asp Gln Ile Leu Val Ser Gly Asn Tyr Lys Ser
260 265 270
Pro Gln Thr Val Lys Ala Leu Asn Val Tyr Ile Gln Asp Pro Pro Tyr
275 280 285
Ala Val Asn Gln Asp Tyr Asp Ala Val Ser Phe Ser Arg Arg Leu Gly
290 295 300
Lys Lys Ser Gly Lys Phe Ser Met Lys Ile Asp Lys Lys Glu Leu Glu
305 310 315 320
Gly Leu Asn Asn Asn Glu Phe Arg Ile Ser Leu Met Phe Ile Leu Ala
325 330 335
Asn Gly Leu His Met Gln Lys His Phe Thr Phe His Trp Asp Ala Leu
340 345 350
Gln Asp Tyr Arg Asp Gly Ser Lys Ser
355 360
<210> 12
<211> 371
<212> PRT
<213> 人工序列
<220>
<223> O-糖蛋白特异性内切蛋白酶(LS)
<400> 12
Met Glu Val Thr Val Pro Asp Ala Leu Lys Asp Arg Ile Ala Leu Lys
1 5 10 15
Lys Thr Ala Arg Gln Leu Asn Ile Val Tyr Phe Leu Gly Ser Asp Thr
20 25 30
Glu Pro Val Pro Asp Tyr Glu Arg Arg Leu Ser Glu Leu Leu Leu Tyr
35 40 45
Leu Gln Gln Phe Tyr Gly Lys Glu Met Gln Arg His Gly Tyr Gly Ala
50 55 60
Arg Ser Phe Gly Leu Asp Ile Lys Ser Pro Gly Arg Val Asn Ile Ile
65 70 75 80
Glu Tyr Lys Ala Lys Asn Pro Ala Ala His Tyr Pro Tyr Glu Asn Gly
85 90 95
Gly Gly Trp Lys Ala Ala Gln Glu Leu Asp Glu Phe Phe Lys Ala His
100 105 110
Pro Asp Arg Lys Lys Ser Gln His Thr Leu Ile Ile Met Pro Thr Trp
115 120 125
Asn Asp Glu Lys Asn Gly Pro Asp Asn Pro Gly Gly Val Pro Phe Tyr
130 135 140
Gly Met Gly Arg Asn Cys Phe Ala Leu Asp Tyr Pro Ala Phe Asp Ile
145 150 155 160
Lys His Leu Gly Gln Lys Thr Arg Glu Gly Arg Leu Leu Thr Lys Trp
165 170 175
Tyr Gly Gly Met Ala His Glu Leu Gly His Gly Leu Asn Leu Pro His
180 185 190
Asn His Gln Thr Ala Ser Asp Gly Lys Lys Tyr Gly Thr Ala Leu Met
195 200 205
Gly Ser Gly Asn Tyr Thr Phe Gly Thr Ser Pro Thr Phe Leu Thr Pro
210 215 220
Ala Ser Cys Ala Leu Leu Asp Ala Cys Glu Val Phe Ser Val Thr Pro
225 230 235 240
Ser Gln Gln Phe Tyr Glu Gly Lys Pro Glu Val Glu Val Gly Asp Val
245 250 255
Ala Ile Ser Phe Lys Gly Asp Gln Ile Leu Val Ser Gly Asn Tyr Lys
260 265 270
Ser Pro Gln Thr Val Lys Ala Leu Asn Val Tyr Ile Gln Asp Pro Pro
275 280 285
Tyr Ala Val Asn Gln Asp Tyr Asp Ala Val Ser Phe Ser Arg Arg Leu
290 295 300
Gly Lys Lys Ser Gly Lys Phe Ser Met Lys Ile Asp Lys Lys Glu Leu
305 310 315 320
Glu Gly Leu Asn Asn Asn Glu Phe Arg Ile Ser Leu Met Phe Ile Leu
325 330 335
Ala Asn Gly Leu His Met Gln Lys His Phe Thr Phe His Trp Asp Ala
340 345 350
Leu Gln Asp Tyr Arg Asp Gly Ser Lys Ser Gly Ser Gly His His His
355 360 365
His His His
370
<210> 13
<211> 6198
<212> DNA
<213> 人工序列
<220>
<223> 编码来自口腔链球菌的O-糖苷酶的核苷酸(具有另外的N末端Met和C末端GSGLE-His6标签并去除信号序列和LPXTG细胞壁锚基序的C末端元件)
<400> 13
atggaccaag cgcgtgtggg tagcaccgat aacctgccga gcgagctggc ggatctggac 60
aagaaagcga gcgacgaagg ccacgatttt gacaaagagg cggcggcgca gaacccgggt 120
agcgcggaaa ccaccgaagg tccgcagacc gaggaagagc tgctggcgca agaaaaagag 180
aagagcgaga agccgagcaa cctgccgaaa gaactggagg ataaactgga aaaggcggag 240
gacaacggtc gtgaagtgga taaagaccag ctggcgcaag acaccggcaa gctggtgccg 300
gaggatgttg cgaaaaccac caacggtgaa ctgaactacg gcgcgaccgt taaaattaag 360
accccgagcg gcgagggtag cggtattgtg gttgcgaagg acctggtgct gaccgttagc 420
cacaacttca ttaaggatag ccaggaaggt aatatccgta aagtggttga taacgaccaa 480
ggcgatggtg acatctacag cattagctat ccgggcctgc cggacgttaa gttcagcaag 540
aaagatatca tccactggga ccgtgagggt tacctgaaag gcttcaagaa cgatctggcg 600
ctggtgcgtc tgcgtaccgt tctggaaaac accccggttg aggtgaccaa gaaaccggtg 660
gttaagaaaa ttggtgacaa gctgcacgtg tttggttatc cggagggcaa actgaacccg 720
atcgtgaaca ccaccgttga tttcgcggaa ccgtacggcg agggtgttca gggcattggt 780
tatcaaggtg gcaaaccggg cgcgagcggt ggcggtatct ttgacaccga aggcaagctg 840
gttggcgtgc accagaacgg tgtggttggc aaacgtagcg gcggtattct gttcagcccg 900
gcgcaactga agtggattca ggaccacatg caaggtatca gcagcgtgaa accggcggat 960
ctggaagaga aagagaagcc ggcggaagag aaaccgaagg aagacaagcc ggcggcggcg 1020
aagccggaaa ccccgaaagc ggttaccccg gagtggcaaa ccgtggcgaa caaggaacag 1080
caaggtaccg ttaccatccg tgaagagaaa ggcgtgcgtt acaaccagct gagcagcacc 1140
gcgcaaaacg ataacgacgg caagccggcg ctgtttgaga aacagggtct gaccgttgac 1200
gcgaacggca acgcgaccgt ggatctgacc ttcaaggacg atagcgaaaa aggcaagagc 1260
cgtttcggcg tttttctgaa attcaaggac accaaaaaca acgtttttgt gggttacgat 1320
caaggcggtt ggttctggga gtataagacc ccgggtaaca gcacctggta caagggtaac 1380
cgtgtggcgg cgccggaacc gggtagcgtg aaccgtctga gcattaccct gaaaagcgac 1440
ggccagctga acgcgagcaa caacgatgtg aacctgttcg acaccgttac cctgccgggt 1500
gcggtgaacg aaaacctgaa gaacgagaag aaaatcctgc tgaaagcggg cacctacagc 1560
aacgaccgta ccgtggttag cgttaagacc gataaccagg aaggtgtgaa agcggacgat 1620
accccggcgc aaaaggaaac cggtccggcg gtggacgata gcaaggttac ctacgacacc 1680
attcagagca aagtgctgaa ggcggttatc gatcaagcgt ttccgcgtgt gaaagagtat 1740
accctgaacg gtcacaccct gccgggtcag gttcagcaat ttaaccaagt gttcattaac 1800
aaccaccgta tcaccccgga agtgacctat aagaaaatta acgaaaccac cgcggagtac 1860
ctgatgaagc tgcgtgacga tgcgcacctg atcaacgcgg aaatgaccgt gcgtctgcag 1920
gtggttgata accaactgca cttcgacgtg accaaaattg ttaaccacaa ccaggttacc 1980
ccgggtcaaa agattgacga tgagcgtaaa ctgctgagca ccatcagctt tctgggcaac 2040
gcgctggtta gcgtgagcag cgatcaagcg ggtgcgaagt ttgatggtgc gaccatgagc 2100
aacaacaccc acgttagcgg tgacgatcac atcgatgtga ccaacccgat gaaagacctg 2160
gcgaagggtt acatgtatgg ctttgttagc accgacaagc tggcggcggg tgtgtggagc 2220
aacagccaaa acagctacgg cggtggcagc aacgattgga cccgtctgac cgcgtataaa 2280
gaaaccgttg gtaacgcgaa ctacgtgggc attcacagca gcgaatggca gtgggagaaa 2340
gcgtacaagg gtatcgtgtt cccggaatat accaaggagc tgccgagcgc gaaagtggtt 2400
atcaccgagg atgcgaacgc ggacaacaaa gtggattggc aggacggtgc gattgcgtac 2460
cgtagcatca tgaacaaccc gcaaggctgg gaaaaagtta aggacattac cgcgtatcgt 2520
atcgcgatga actttggtag ccaggcgcaa aacccgttcc tgatgaccct ggacggcatc 2580
aagaaaatta acctgcacac cgatggcctg ggtcagggcg ttctgctgaa gggttatggt 2640
agcgagggtc atgacagcgg tcacctgaac tacgcggata tcggtaaacg tattggtggc 2700
gtggaagact ttaagaccct gattgagaaa gcgaagaaat acggtgcgca cctgggcatc 2760
cacgttaacg cgagcgaaac ctacccggag agcaagtatt tcaacgaaaa cattctgcgt 2820
aaaaacccgg acggtagcta cagctatggc tggaactggc tggatcaggg tatcaacatt 2880
gatgcggcgt acgacctggc gcatggccgt ctggcgcgtt gggaggacct gaagaaaaag 2940
ctgggtgaag gcctggattt tatctatgtt gacgtgtggg gtaacggtca gagcggtgat 3000
aacggtgcgt gggcgaccca tgtgctggcg aaagagatta acaagcaagg ttggcgtttt 3060
gcgatcgaat ggggccacgg tggcgagtac gacagcacct tccagcactg ggcggcggat 3120
ctgacctacg gtggctatac caacaagggt atcaacagcg cgattacccg tttcatccgt 3180
aaccaccaga aagatagctg ggttggcgac taccgtagct atggtggcgc ggcgaactac 3240
ccgctgctgg gtggctatag catgaaggac tttgagggtt ggcaaggccg tagcgattac 3300
aacggttatg ttaccaacct gttcgcgcac gacgtgatga ccaagtactt tcagcacttc 3360
accgttagca aatgggaaaa cggtaccccg gtgaccatga ccgataacgg cagcacctat 3420
aagtggaccc cggaaatgaa agtggagctg gttgacgcgg cgggtaacaa ggtggttgtg 3480
acccgtaaaa gcaacgatgt gaacagcccg cagtaccgtg agcgtaccgt taccctgaac 3540
ggtcgtgtga tccaagacgg cagcgcgtat ctgaccccgt ggaactggga tgcgaacggt 3600
aaaaagctgc cgaccgaaaa agagaagatg tactatttta acacccaagc gggtgcgacc 3660
acctggaccc tgccgagcga ctgggcgaac agcaaggttt acctgtataa actgaccgat 3720
cagggcaaga ccgaggagca agaactgacc gtgaccgatg gcaaaattac cctggacctg 3780
ctggcgaacc agccgtacgt tctgtatcgt agcaagcaaa ccaacccgga aatgagctgg 3840
agcgagggta tgcacatcta cgaccaaggt ttcaacagcg gcaccctgaa acactggacc 3900
attagcggcg atgcgagcaa ggcggagatc gtgaaaagcc agggtgcgaa cgaaatgctg 3960
cgtatccaag gcaacaaaag caaggttagc ctgacccaga agctgaccgg tctgaaaccg 4020
aacaccaagt acgcggttta tgtgggcgtt gacaaccgta gcaacgcgaa agcgagcatt 4080
accgttaaca ccggtgaaaa agaggtgacc acctacacca acaagagcct ggcgctgaac 4140
tacatcaaag cgtatgcgca caacaaccgt cgtgagaacg cgaccgtgga cgataccagc 4200
tacttccaga acatgtatgc gttctttacc accggtagcg acgtgagcaa cgttaccctg 4260
accctgagcc gtgaagcggg cgatgaggcg acctattttg acgaaattcg taccttcgag 4320
aacaacagca gcatgtacgg tgataagcac gacaccggtc agggcacctt taaacaagat 4380
ttcgaaaacg ttgcgcaagg tatcttcccg tttgttgtgg gtggcgtgga aggcgttgag 4440
gacaaccgta cccacctgag cgagaagcac gatccgtaca cccagcgtgg ttggaacggc 4500
aaaaaggtgg acgatgttat tgagggtaac tggagcctga aaaccaacgg cctggttagc 4560
cgtcgtaacc tggtgtacca gaccatcccg caaaacttcc gttttgaggc gggcaagacc 4620
taccgtgtga cctttgaata tgaggcgggc agcgacaaca cctatgcgtt tgttgtgggt 4680
aaaggcgaat tccagagcgg tcgtcgtggc acccaagcga gcaacctgga aatgcacgag 4740
ctgccgaaca cctggaccga tagcaaaaag gcgaaaaagg tgaccttcct ggttaccggt 4800
gcggaaaccg gtgacacctg ggtgggtatc tacagcaccg gcaacgcgag caacacccgt 4860
ggtgatgcgg gtggcaacgc gaactttcgt ggctataacg atttcatgat ggacaacctg 4920
caaatcgaag agattaccct gaccggcaag atgctgaccg aaaacgcgct gaaaaactat 4980
ctgccgaccg ttgcgatgac caactacacc aaggaaagca tggacgcgct gaaagaggcg 5040
gttttcaacc tgagccaggc ggacgatgac atcagcgtgg aagaggcgcg tgcggaaatc 5100
gcgaagattg aggcgctgaa aaacgcgctg gttcagaaaa agaccgcgct ggttgcggaa 5160
gattttgaga gcctggatgc gccggcgcaa ccgggtgaag gcctggagaa cgcgttcgac 5220
ggtaacgtta gcagcctgtg gcacaccagc tggaacggtg gcgatgttgg caagccggcg 5280
accatggtgc tgaaagaacc gaccgagatc accggtctgc gttatgtgcc gcgtgcgagc 5340
gatagcaacg gcaacctgcg tgacgttaag ctggttgtga ccgatgaaag cggtaaagag 5400
cacaccttta acgtgaccga ctggccgaac aacaacaaac cgaaggatat tgacttcggc 5460
aaaaccatta aggcgaaaaa gatcgttctg accggtacca agacctacgg cgatggtggc 5520
gacaaatatc agagcgcggc ggagctgatc tttacccgtc cgcaagtggc ggaaaccccg 5580
ctggatctga gcggttacga agcggcgctg gcgaaagcgc agaagctgac cgataaggac 5640
aaccaggaag aggtggcgag cgttcaagcg agcatgaaat atgcgaccga caaccacctg 5700
ctgaccgaac gtatggttgc gtacttcgcg gattatctga accaactgaa ggatagcgcg 5760
accaaaccgg atgcgccgac cagcagcaag ggtgaagaac agccgccggt gctggatgtt 5820
ccggagttta aaggtggcgt gaacgcgacc gaggcggcgg tgcacgaagt tccggagttc 5880
aagggtggcg tgaacgcggt tcaggcgctg gttcacgaac tgccggagta taaaggtggc 5940
gcgaacgcgg ttctggcggc ggcgaacgaa gtgccggagt acaagggtgg cgcgaacgcg 6000
gtggaagcgc tggttaacga gaaaccggcg tataccggtg ttctggcgac cgcgggcgac 6060
caggcggcgc cgaccgtgga aaaaccggag tacccgctga ccccgagccc ggttgcggac 6120
accaaaaccc cgggtgcgaa agatgaagag aagctgccgg cgggtagcgg cctcgagcac 6180
caccaccacc accactga 6198

Claims (11)

1.一种组合物或组合,所述组合物或组合包含第一唾液酸酶、第二唾液酸酶和O-糖苷酶:其中:
(a)所述第一唾液酸酶是由SEQ ID NO: 1、SEQ ID NO: 2或SEQ ID NO: 3中的任何一个的氨基酸序列组成的多肽;
(b)所述第二唾液酸酶是由SEQ ID NO: 4、SEQ ID NO: 5或SEQ ID NO: 6中的任何一个的氨基酸序列组成的多肽;并且
(c)所述O-糖苷酶是由SEQ ID NO: 7、SEQ ID NO: 8、SEQ ID NO: 9或SEQ ID NO: 10中的任何一个的氨基酸序列组成的多肽。
2.根据权利要求1所述的组合物或组合,其特征在于,所述组合物能够水解糖蛋白中的>90%的唾液酸键。
3. 根据权利要求1或2所述的组合物或组合,其特征在于,所述组合物或组合包含:
(a)由SEQ ID NO: 3氨基酸序列组成的唾液酸酶和由SEQ ID NO: 6氨基酸序列组成的唾液酸酶;和/或
(b)比例为1:1的由SEQ ID NO: 3氨基酸序列组成的唾液酸酶和由SEQ ID NO: 6氨基酸序列组成的唾液酸酶。
4.根据权利要求1或2所述的组合物或组合,其特征在于,所述组合物中不多于两种多肽是自嗜粘蛋白艾克曼菌获得的唾液酸酶。
5.根据权利要求1或2所述的组合物或组合,其特征在于,所述组合物另外包含O-糖苷酶特异性内切蛋白酶。
6.根据权利要求5所述的组合物或组合,其特征在于,所述第一唾液酸酶、所述第二唾液酸酶、所述O-糖苷酶和/或所述O-糖苷酶特异性内切蛋白酶以高度纯化或分离的形式存在。
7. 根据权利要求5所述的组合物或组合,其特征在于,所述O-糖苷酶特异性内切蛋白酶是选自以下的O-糖蛋白特异性内切蛋白酶:
a.由SEQ ID NO: 11氨基酸序列组成的多肽;或
b. 由SEQ ID NO: 12氨基酸序列组成的多肽。
8.一种修饰糖蛋白的方法,所述方法包括使包含所述糖蛋白的样品与根据前述权利要求1至7中任一项所述的组合物或组合接触。
9.根据权利要求8所述的方法,其特征在于,对所得产物进行分析并且其中所述分析包括通过任何合适的手段来分开和/或检测和/或分离所述产物,所述合适的手段包括SDS-PAGE、HPLC、凝集素印迹、ELISA或质谱。
10.根据权利要求8或9所述的方法,其特征在于,所述组合物或组合如权利要求1至4和6中任一项所述,并且其中所述方法包括在所述糖蛋白与所述组合物或组合接触的同时、之前或之后使所述样品与O-糖苷酶特异性内切蛋白酶接触。
11.根据权利要求8所述的方法,其特征在于,所述组合物或组合另外包含O-糖苷酶特异性内切蛋白酶,其中所述O-糖苷酶特异性内切蛋白酶如权利要求7所定义。
CN201880050240.9A 2017-05-26 2018-05-25 用于聚糖分析的工具 Active CN111183220B (zh)

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GBGB1708471.6A GB201708471D0 (en) 2017-05-26 2017-05-26 Polypeptide
GB1708476.5 2017-05-26
GBGB1708476.5A GB201708476D0 (en) 2017-05-26 2017-05-26 Tools for glycan analysis
GB1708471.6 2017-05-26
GB1806655.5 2018-04-24
GBGB1806655.5A GB201806655D0 (en) 2018-04-24 2018-04-24 Polypeptide
PCT/EP2018/063833 WO2018215657A1 (en) 2017-05-26 2018-05-25 Enzymes for glycan analysis

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