CN114410602B - 一种末端脱氧核苷酸转移酶的突变体及其应用 - Google Patents
一种末端脱氧核苷酸转移酶的突变体及其应用 Download PDFInfo
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- CN114410602B CN114410602B CN202210103671.5A CN202210103671A CN114410602B CN 114410602 B CN114410602 B CN 114410602B CN 202210103671 A CN202210103671 A CN 202210103671A CN 114410602 B CN114410602 B CN 114410602B
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- C12Y—ENZYMES
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Abstract
本发明公开一种末端脱氧核苷酸转移酶的突变体,其特征在于:包含如SEQ ID NO:2中列出的氨基酸序列或功能等同序列,以及至少在对应于残基P215的位置处的一个氨基酸取代或功能等同残基,其中该位置参照SEQ ID NO:2所示的氨基酸序列编号;所述突变体能够在没有模板的情况下合成核酸片段;所述突变体能够将修饰的核苷酸掺入到核酸片段中。
Description
技术领域
本发明涉及一种末端脱氧核苷酸转移酶的突变体及其应用,属于基因工程、酶工程领域,具体涉及一种末端脱氧核苷酸转移酶、其编码基因及其表达和应用。
背景技术
末端脱氧核苷酸转移酶(TdT)是遗传工程中的重要工具酶之一,是一种特殊的DNA聚合酶,可以在没有模板存在的条件下催化脱氧核苷酸结合到DNA分子的3'羟基端。此外,带有突出、凹陷或平滑末端的单双链DNA分子均可作为TdT的底物。
末端转移酶在分子生物学中应用广泛,例如,该酶可用于cDNA末端的快速扩增(RACE)中添加核苷酸,并可以用来作为在后续PCR的引物的模板;在检测细胞凋亡常用的TUNEL检测中,该酶也可以用于添加标记放射性同位素的核苷酸;在基因测序中,可使用该酶在3’末端加双脱氧核苷酸封端,以此降低测序反应的杂信号的产生。
小牛胸腺TdT是一种常用的类型,目前野生型末端转移酶以非天然脱氧核糖核苷酸如ddNTP为底物时封端效率较低,反应时间过长,且在37℃持续工作时衰减较快。通过对蛋白结构分析,突变部分位点,提高TdT末端转移酶的活性以及持续工作能力显得尤为重要,因此需要开发一种高活性和高稳定性的末端脱氧核苷酸转移酶。
发明内容
本发明提供一种小牛胸腺末端脱氧核苷酸转移酶的突变体,通过突变关键位点的氨基酸改变氨基酸的带电性或疏水性,从而改变酶的空间结构,使其可以更好地与DNA结合,从而提高末端转移酶3’OH末端的催化能力,该突变体具有更高的酶活性,且在37℃保存下该突变体的稳定性更高。
具体的,本发明提供一种末端脱氧核苷酸转移酶的突变体,其特征在于:
包含如SEQ ID NO:2中列出的氨基酸序列或功能等同序列,以及至少在对应于残基P215的位置处的一个氨基酸取代或功能等同残基,其中该位置参照SEQ ID NO:2所示的氨基酸序列编号;
所述突变体能够在没有模板的情况下合成核酸片段;
所述突变体能够将修饰的核苷酸掺入到核酸片段中。
根据优选的实施方式,所述修饰的核苷酸包括3’O-修饰的核苷酸,更优选地,包括3’O-封闭的核苷酸。
根据优选的实施方式,所述取代选自P215D、P215E、P215V。
根据优选的实施方式,所述突变体还在对应于选自F267和G372的残基的位置处包含至少一个氨基酸取代或功能等同残基。
根据优选的实施方式,所述突变体在对应于选自P215、F267和G372的残基的位置处包含至少两个氨基酸取代,优选地包含三个氨基酸取代。
根据优选的实施方式,所述取代选自P215D/E/V、F267A/G/S、G372I/Q/T。
根据优选的实施方式,所述突变体在SEQ ID NO:2或功能等同序列的N末端和/或C末端处包含标签序列,包括组氨酸标签序列。
本发明还提供一种核酸分子,所述核酸分子编码如前面所述任一项中定义的末端脱氧核苷酸转移酶的突变体。
本发明还提供一种表达载体,所述表达载体中包含前述的核酸分子序列。
本发明还提供一种宿主细胞,所述宿主细胞包含前述的核酸分子或前述的表达载体。
本发明还提供一种用于产生本发明中所定义的末端脱氧核苷酸转移酶的突变体的方法,使本发明中所述宿主细胞在允许编码所述突变体的核酸分子表达的培养条件下被培养,并且其中所述突变体可被回收,优选的,并进行纯化。
根据优选的实施方式,本发明还提供将所定义的末端脱氧核苷酸转移酶的突变体用于在没有模板的情况下用3’O-修饰的核苷酸合成核酸分子的用途。
本发明还提供一种在没有模板的情况下合成核酸分子的方法,所述方法包括使核酸引物与至少一种核苷酸,优选地至少一种3’O-修饰的核苷酸与本发明中任一项中所定义的末端脱氧核苷酸转移酶的突变体接触的步骤。
本发明还提供一种用于进行核苷酸掺入反应的试剂盒,所述试剂盒包含如前所述任一项中所定义的末端脱氧核苷酸转移酶的突变体,缓冲液,一种或更多种核苷酸,优选地一种或更多种3’O-修饰的核苷酸,以及任选地至少一种核酸引物。
本发明的有益效果
本发明提供一种来自小牛胸腺的末端脱氧核苷酸转移酶(TdT)的突变体,相比于野生型酶,该突变体具有如下优势:
1.酶活性明显提高,增加了3-5倍;
2.在稳定性上,该突变体相比于野生型末端转移酶以及NEB公司的商品化末端转移酶表现出更高的稳定性。
附图说明
图1.纯化后的TdT末端转移酶突变体的凝胶电泳图。SDS-PAGE,M代表marker,箭头所示条带为末端转移酶,大小约为58kDa。
图2.不同末端转移酶的相对活性结果图。
图3.不同末端转移酶的热稳定性检测结果图。
具体实施方式
下面结合实施例对本发明作进一步的说明。然而,本发明的范围并不限于下述实施例。本领域的技术人员能够理解,在不背离本发明的精神和范围的前提下,可以对本发明进行各种变化和修饰。
本发明对试验中所使用到的材料以及试验方法进行一般性和/或具体的描述。虽然为实现本发明目的所使用的许多材料和操作方法是本领域公知的,但是本发明仍然在此作尽可能详细的描述。
下述实施例中所涉及的仪器、试剂、材料等,若无特别说明,均为现有技术中已有的常规仪器、试剂、材料等,可通过正规商业途径获得。下述实施例中所涉及的实验方法、检测方法等,若无特别说明,均为现有技术中已有的常规实验方法、检测方法等。
除非另外定义,否则本文使用的所有科学和技术术语的含义与本领域普通技术人员通常理解的含义相同。
本发明提供一种小牛胸腺末端脱氧核苷酸转移酶(简称为TdT)的突变体,与野生型TdT相比,该突变体具有更高的酶活性,且在37℃保存该突变体的稳定性更高。
具体的,本发明提供一种末端脱氧核苷酸转移酶的突变体,其特征在于:
包含如SEQ ID NO:2中列出的氨基酸序列或功能等同序列,以及至少在对应于残基P215的位置处的一个氨基酸取代或功能等同残基,其中该位置参照SEQ ID NO:2所示的氨基酸序列编号;
所述突变体能够在没有模板的情况下合成核酸片段;
所述突变体能够将修饰的核苷酸掺入到核酸片段中。
本发明中,野生型小牛胸腺末端脱氧核苷酸转移酶氨基酸序列如下所示(5’→3’),如SEQ ID NO:2所示:
MDPLCTASSGPRKKRPRQVGASMASPPHDIKFQNLVLFILEKKMGTTRRNFLMELARRKGFRVENELSDSVTHIVAENNSGSEVLEWLQVQNIRASSQLELLDVSWLIESMGAGKPVEITGKHQLVVRTDYSATPNPGFQKTPPLAVKKISQYACQRKTTLNNYNHIFTDAFEILAENSEFKENEVSYVTFMRAASVLKSLPFTIISMKDTEGIPCLGDKVKCIIEEIIEDGESSEVKAVLNDERYQSFKLFTSVFGVGLKTSEKWFRMGFRSLSKIMSDKTLKFTKMQKAGFLYYEDLVSCVTRAEAEAVGVLVKEAVWAFLPDAFVTMTGGFRRGKKIGHDVDFLITSPGSAEDEEQLLPKVINLWEKKGLLLYYDLVESTFEKFKLPSRQVDTLDHFQKCFLILKLHHQRVDSSKSNQQEGKTWKAIRVDLVMCPYENRAFALLGWTGSRQFERDIRRYATHERKMMLDNHALYDKTKRVFLKAESEEEIFAHLGLDYIEPWERNA
编码上述野生型小牛胸腺末端脱氧核苷酸转移酶的基因序列如下所示(5’→3’),如SEQ ID NO:1所示:
ATGGATCCGCTGTGCACAGCCTCCTCAGGCCCTCGGAAGAAGAGACCCAGGCAGGTGGGTGCCTCAATGGCCTCCCCTCCTCATGACATCAAGTTTCAAAATTTGGTCCTCTTCATTTTGGAGAAGAAAATGGGAACCACCCGCAGAAACTTCCTCATGGAGCTGGCTCGAAGGAAAGGTTTCAGGGTTGAAAATGAGCTCAGTGATTCTGTCACCCACATTGTAGCAGAAAACAACTCTGGTTCAGAGGTTCTCGAGTGGCTTCAGGTACAGAACATAAGAGCCAGCTCGCAGCTAGAACTCCTTGATGTCTCCTGGCTGATCGAAAGTATGGGAGCAGGAAAACCAGTGGAGATTACAGGAAAACACCAGCTTGTTGTGAGAACAGACTATTCAGCTACCCCAAACCCAGGCTTCCAGAAGACTCCACCACTTGCTGTAAAAAAGATCTCCCAGTACGCGTGTCAAAGAAAAACCACTTTGAACAACTATAACCACATATTCACGGATGCCTTTGAGATACTGGCTGAAAATTCTGAGTTTAAAGAAAATGAAGTCTCTTATGTGACATTTATGAGAGCAGCTTCTGTACTTAAATCTCTGCCATTCACAATCATCAGTATGAAGGATACAGAAGGAATTCCCTGCCTGGGGGACAAGGTGAAGTGTATCATAGAGGAAATTATTGAAGATGGAGAAAGTTCTGAAGTTAAAGCTGTGTTAAATGATGAACGATATCAGTCCTTCAAACTCTTTACTTCTGTTTTTGGAGTGGGACTGAAGACATCTGAGAAATGGTTCAGGATGGGGTTCAGATCTCTGAGTAAAATAATGTCAGACAAAACCCTGAAATTCACAAAAATGCAGAAAGCAGGATTTCTCTATTATGAAGACCTTGTCAGCTGCGTGACCAGGGCCGAAGCAGAGGCGGTTGGCGTGCTGGTTAAAGAGGCTGTGTGGGCATTTCTGCCGGATGCCTTTGTCACCATGACAGGAGGATTCCGCAGGGGTAAGAAGATTGGGCATGATGTAGATTTTTTAATTACCAGCCCAGGATCAGCAGAGGATGAAGAGCAACTTTTGCCTAAAGTGATAAACTTATGGGAAAAAAAGGGATTACTTTTATATTATGACCTTGTGGAGTCAACATTTGAAAAGTTCAAGTTGCCAAGCAGGCAGGTGGATACTTTAGATCATTTTCAAAAATGCTTTCTGATTTTAAAATTGCACCATCAGAGAGTAGACAGTAGCAAGTCCAACCAGCAGGAAGGAAAGACCTGGAAGGCCATCCGTGTGGACCTGGTTATGTGCCCCTACGAGAACCGTGCCTTTGCCCTGCTAGGCTGGACTGGCTCCCGGCAGTTTGAGAGAGACATCCGGCGCTATGCCACACACGAGCGGAAGATGATGCTGGATAACCACGCTTTATATGACAAGACCAAGAGGGTATTTCTCAAAGCGGAAAGTGAAGAAGAAATCTTTGCACATCTGGGATTGGACTACATTGAACCATGGGAAAGAAATGCTTAG
在本发明中,“功能等同序列”指的是与SEQ ID NO:2同源的TdT的序列。“功能等同残基”意指在与SEQ ID NO:2序列同源的TdT的序列中的并且具有相同功能的残基。
在本发明中,“取代”指的是一个氨基酸残基被另一个氨基酸残基替代。以下术语用于表示取代,例如,P215E表示野生型序列的位置215处的氨基酸残基(脯氨酸,P)被改变为谷氨酸(E)。P215 D/E/V表示野生型序列的位置215处的氨基酸残基(脯氨酸,P)被以下氨基酸之一取代:天冬氨酸(D)、谷氨酸(E)或缬氨酸(V)。
根据优选的实施方式,所述残基P215的位置处的一个氨基酸取代选自P215D、P215E、P215V。
根据优选的实施方式,所述突变体在对应于选自F267和G372的残基的位置处包含至少一个氨基酸取代或功能等同残基。即所述突变体的突变残基是P215、F267;或者P215、G372;或者P215、F267、G372。
根据优选的实施方式,所述突变体在对应于选自P215、F267和G372的残基的位置处包含至少两个氨基酸取代,优选地包含三个氨基酸取代。
根据优选的实施方式,所述取代选自P215D/E/V、F267A/G/S、G372I/Q/T。所述突变体包含选自以下的三个氨基酸取代的组合:P215D+F267A+G372I,P215D+F267A+G372Q,P215D+F267A+G372T,P215D+F267G+G372I,P215D+F267G+G372Q,P215D+F267G+G372T,P215D+F267S+G372I,P215D+F267S+G372Q,P215D+F267S+G372T,P215E+F267A+G372I,P215E+F267A+G372Q,P215E+F267A+G372T,P215E+F267G+G372I,P215E+F267G+G372Q,P215E+F267G+G372T,P215E+F267S+G372I,P215E+F267S+G372Q,P215E+F267S+G372T,P215V+F267A+G372I,P215V+F267A+G372Q,P215V+F267A+G372T,P215V+F267G+G372I,P215V+F267G+G372Q,P215V+F267G+G372T,P215V+F267S+G372I,P215V+F267S+G372Q,P215V+F267S+G372T。
根据优选的实施方式,所述突变体在SEQ ID NO:2或功能等同序列的N末端和/或C末端处包含标签序列,包括组氨酸标签序列。His-Tag(组氨酸标签)由6-10个组氨酸残基组成,分子量较小,通常插入在目的蛋白的C末端或N末端,是目前原核表达最常用的标签。应该理解的是,本发明所述标签不限于His-Tag,文献中已经报道了不同的标签,因此本发明涵盖了技术人员已知的所有适合的标签。
本发明还提供一种核酸分子,所述核酸分子编码如前面所述任一项中定义的末端脱氧核苷酸转移酶的突变体。
本发明还提供一种表达载体,所述表达载体中包含前述的核酸分子序列,包括但不限于pET-21b表达载体。
本发明还提供一种宿主细胞,所述宿主细胞包含前述的核酸分子或前述的表达载体。
本发明还提供一种用于产生本发明中所定义的TdT的突变体的方法,使本发明中所述宿主细胞在允许编码所述突变体的核酸分子表达的培养条件下被培养,并且其中所述突变体可被回收,优选的,并进行纯化。
根据优选的实施方式,本发明还提供将所定义的TdT的突变体用于在没有模板的情况下用3’O-修饰的核苷酸合成核酸分子的用途。
本发明还提供一种在没有模板的情况下合成核酸分子的方法,所述方法包括使核酸引物与至少一种核苷酸,优选地至少一种3’O-修饰的核苷酸与本发明中任一项中所定义的TdT的突变体接触的步骤。
本发明还提供一种用于进行核苷酸掺入反应的试剂盒,所述试剂盒包含如前所述任一项中所定义的TdT的突变体,缓冲液,一种或更多种核苷酸,优选地一种或更多种3’O-修饰的核苷酸,以及任选地至少一种核酸引物。
根据优选的实施方式,本发明中所述修饰的核苷酸包括3’O-修饰的核苷酸,更优选地,包括3’O-封闭的核苷酸;包括,例如,在3’末端具有附加基团,所述附加基团阻止了核苷酸的进一步添加,即通过用保护基取代3’-OH基防止了核苷酸的进一步添加。在进行测序反应的核酸链的延伸过程中,经常会利用此封端作用来降低测序杂反应的发生。
根据优选的实施方式,本发明方法使用可逆的3’-封端基团,所述可逆的3’-封端基团可通过裂解去除以允许添加另外的核苷酸。
本发明以野生型小牛胸腺末端脱氧核苷酸转移酶的基因序列为模板,通过PCR以及分子克隆技术改变所述基因的碱基序列,从而实现氨基酸序列的定点突变。主要包括以下步骤:
1)根据突变位点设计引物,通过PCR扩增将突变位点的序列改变,并拼接出全长基因序列。
2)将拼接的全长基因序列用NdeI和XhoI进行双酶切,通过T4连接酶克隆到pET-21b载体上。
3)将上述连接产物转化到DH5α感受态细胞中,通过涂布氨苄抗性的平板培养。
4)通过PCR筛选阳性克隆,并通过测序验证突变体。
5)将测序验证为目标突变体的阳性克隆转化到BL21感受态细胞中,诱导表达。
6)破碎诱导后的细胞,通过亲和层析色谱和离子交换色谱纯化目的蛋白。
具体的步骤可参见实施例1。
实施例1
1.构建载体
1)根据SEQ ID NO:1所示序列进行基因序列合成,并将所合成的全基因片段插入载体pET-21b(NdeI和XhoI)中。本实施例中使用的是pET-21b表达载体,但不限于此载体。
2)根据突变点P215、F267、G372的位置,修改DNA序列使其对应的氨基酸发生改变,可修改为P215D、P215E、P215V、F267A、F267G、F267S、G372 I、G372Q、G372T,不局限于其中的一种或多种突变。
2.诱导表达
1)从氨苄抗性的LB固体培养基上挑取单克隆菌落,置于150mL LB液体培养基中,该培养基中含有150μl 100mg/ml氨苄霉素,将此培养瓶置于恒温振动培养箱中以200rpm转速、37℃培养16h。
2)取10ml培养好的菌种接种于1L/瓶LB液体培养基中,并加入1ml100mg/ml氨苄霉素,用封口膜封闭瓶口,置于恒温振动培养箱中以200rpm转速、37℃培养约4h。
3)从1L/瓶LB液体培养菌液中取1ml菌液,用分光光度计监测菌液在600nm波长时的吸收值,当吸收值在0.8-1.0之间时向每瓶菌液加入1ml 1MIPTG,在恒温振荡培养箱中以200rpm转速、16℃继续培养16h。
3.TdT末端转移酶的纯化
1)称取TdT酶湿菌20g放入200ml玻璃烧杯中。
2)用量桶量取100ml Lysis buffer(TdT-Lysis buffer:50mM KH2PO4,5%甘油pH6.0
)倒入烧杯中,磁力搅拌器搅拌至无块状菌体。
3)将重悬菌液缓慢倒入高压均质机进样杯中,设置速度:50;压力800-1000bar;开始破碎,接收破碎后液体,12000rpm 4℃离心15min。
4)将上清转移至干净烧杯中,用0.22μm滤膜过滤。
5)用Lysis buffer平衡SP-HP层析柱后,将上清上柱。
6)上样结束后再次平衡至UV检测值趋于平衡。
7)用Elution buffer(50mM KH2PO4,5%甘油,1M NaCl,pH 6.0)线性梯度洗脱15个柱体积。
8)将洗脱下来的峰收集,过镍柱亲和层析。
9)用binding buffer平衡镍柱亲和层析柱。
10)平衡后将过SP柱收集的蛋白,上样镍柱亲和层析柱。
11)用Elution buffer(50mM KH2PO4、5%甘油、0.5M NaCl、250mM咪唑pH 7.6)线性梯度洗脱15个柱体积。
12)将镍柱洗脱出来的峰过凝胶过滤柱。
实施例2
以NEB TdT末端转移酶(货号:M0315L)为对照,用本方法对其进行活性检测。
1.取bio-rad白管黑托酶标板一块,将配制的反应液分到酶标板中,
每孔45μL。
反应体系如下:
组分 | 体积/μL |
primer(5uM) | 2 |
dATP-dTTP(5mM each) | 5 |
CoCl2(2.5mM) | 5 |
Buffer(10×) | 5 |
H2O | 28 |
Total | 45 |
2.用移液器取待测样品5ul加入到酶标板的反应液中,吹吸2次混匀。
3.用透明密封模封住酶标板,将酶标板放在PCR仪上,设置热盖80℃,反应温度37℃2.5h,70℃10min,4℃保存。
4.待反应结束后,取出酶标板撕开封口膜。
5.新鲜配制铜络合液,振荡混匀:
组分 | 体积/μL |
(+)-L-抗坏血酸钠(100mM) | 20 |
CuSO4溶液(10mM) | 5 |
NaCl溶液(150mM) | 100 |
Total | 125 |
6.取步骤5配制的铜络合液125ul加入到步骤4的酶标板中,吹吸2次混匀,静置5min。
7.酶标仪设置激发光340nm,发射光570nm,读数。
8.分析:具体实验结果见图2,NEB(M0315L)与野生型相比,二者的酶活接近,本发明中的三个TdT突变体的酶活明显升高3-4倍。
酶活定义:一单位是指在检测条件下使用d(pT)6作为引物,在+37℃时30分钟内将1nMol dTMP掺入酸性不溶性产物中的酶活。
单位测定条件:在120μl的反应体积中,200mM二甲胂酸钾、1mM CoCl2、1mM dTTP、0.1OD d(pT)6、6.25pmol3H dTTP。
实施例3
对不同TdT突变体进行稳定性检测。
根据实施例2,将不同的TdT末端转移酶调整到相同活性浓度条件下(即20U/ul),将酶放在37℃的环境中,分别在3天、6天、9天、12天、15天后对其进行活性检测,结果如图3所示。
从图3中可以看出,NEB(M0315L)与野生型的TdT的活性变化趋势接近,随着时间推移,二者的酶活均快速下降;而本发明的三种酶突变体,放置6天后开始直到15天,活性明显高于野生型及NEB(M0315L),且随着时间推移,这种优势更加明显,即本发明的TdT突变体的稳定性更高。
序列表
<110> 赛纳生物科技(北京)有限公司
<120> 一种末端脱氧核苷酸转移酶的突变体及其应用
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1530
<212> DNA
<213> Bos taurus
<400> 1
atggatccgc tgtgcacagc ctcctcaggc cctcggaaga agagacccag gcaggtgggt 60
gcctcaatgg cctcccctcc tcatgacatc aagtttcaaa atttggtcct cttcattttg 120
gagaagaaaa tgggaaccac ccgcagaaac ttcctcatgg agctggctcg aaggaaaggt 180
ttcagggttg aaaatgagct cagtgattct gtcacccaca ttgtagcaga aaacaactct 240
ggttcagagg ttctcgagtg gcttcaggta cagaacataa gagccagctc gcagctagaa 300
ctccttgatg tctcctggct gatcgaaagt atgggagcag gaaaaccagt ggagattaca 360
ggaaaacacc agcttgttgt gagaacagac tattcagcta ccccaaaccc aggcttccag 420
aagactccac cacttgctgt aaaaaagatc tcccagtacg cgtgtcaaag aaaaaccact 480
ttgaacaact ataaccacat attcacggat gcctttgaga tactggctga aaattctgag 540
tttaaagaaa atgaagtctc ttatgtgaca tttatgagag cagcttctgt acttaaatct 600
ctgccattca caatcatcag tatgaaggat acagaaggaa ttccctgcct gggggacaag 660
gtgaagtgta tcatagagga aattattgaa gatggagaaa gttctgaagt taaagctgtg 720
ttaaatgatg aacgatatca gtccttcaaa ctctttactt ctgtttttgg agtgggactg 780
aagacatctg agaaatggtt caggatgggg ttcagatctc tgagtaaaat aatgtcagac 840
aaaaccctga aattcacaaa aatgcagaaa gcaggatttc tctattatga agaccttgtc 900
agctgcgtga ccagggccga agcagaggcg gttggcgtgc tggttaaaga ggctgtgtgg 960
gcatttctgc cggatgcctt tgtcaccatg acaggaggat tccgcagggg taagaagatt 1020
gggcatgatg tagatttttt aattaccagc ccaggatcag cagaggatga agagcaactt 1080
ttgcctaaag tgataaactt atgggaaaaa aagggattac ttttatatta tgaccttgtg 1140
gagtcaacat ttgaaaagtt caagttgcca agcaggcagg tggatacttt agatcatttt 1200
caaaaatgct ttctgatttt aaaattgcac catcagagag tagacagtag caagtccaac 1260
cagcaggaag gaaagacctg gaaggccatc cgtgtggacc tggttatgtg cccctacgag 1320
aaccgtgcct ttgccctgct aggctggact ggctcccggc agtttgagag agacatccgg 1380
cgctatgcca cacacgagcg gaagatgatg ctggataacc acgctttata tgacaagacc 1440
aagagggtat ttctcaaagc ggaaagtgaa gaagaaatct ttgcacatct gggattggac 1500
tacattgaac catgggaaag aaatgcttag 1530
<210> 2
<211> 509
<212> PRT
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Met Asp Pro Leu Cys Thr Ala Ser Ser Gly Pro Arg Lys Lys Arg Pro
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Arg Gln Val Gly Ala Ser Met Ala Ser Pro Pro His Asp Ile Lys Phe
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Gln Asn Leu Val Leu Phe Ile Leu Glu Lys Lys Met Gly Thr Thr Arg
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Arg Asn Phe Leu Met Glu Leu Ala Arg Arg Lys Gly Phe Arg Val Glu
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Asn Glu Leu Ser Asp Ser Val Thr His Ile Val Ala Glu Asn Asn Ser
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Gly Ser Glu Val Leu Glu Trp Leu Gln Val Gln Asn Ile Arg Ala Ser
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Ser Gln Leu Glu Leu Leu Asp Val Ser Trp Leu Ile Glu Ser Met Gly
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Ala Gly Lys Pro Val Glu Ile Thr Gly Lys His Gln Leu Val Val Arg
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Thr Asp Tyr Ser Ala Thr Pro Asn Pro Gly Phe Gln Lys Thr Pro Pro
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Leu Ala Val Lys Lys Ile Ser Gln Tyr Ala Cys Gln Arg Lys Thr Thr
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Leu Asn Asn Tyr Asn His Ile Phe Thr Asp Ala Phe Glu Ile Leu Ala
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Glu Asn Ser Glu Phe Lys Glu Asn Glu Val Ser Tyr Val Thr Phe Met
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Arg Ala Ala Ser Val Leu Lys Ser Leu Pro Phe Thr Ile Ile Ser Met
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Lys Asp Thr Glu Gly Ile Pro Cys Leu Gly Asp Lys Val Lys Cys Ile
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Ile Glu Glu Ile Ile Glu Asp Gly Glu Ser Ser Glu Val Lys Ala Val
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Leu Asn Asp Glu Arg Tyr Gln Ser Phe Lys Leu Phe Thr Ser Val Phe
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Gly Val Gly Leu Lys Thr Ser Glu Lys Trp Phe Arg Met Gly Phe Arg
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Ser Leu Ser Lys Ile Met Ser Asp Lys Thr Leu Lys Phe Thr Lys Met
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Gln Lys Ala Gly Phe Leu Tyr Tyr Glu Asp Leu Val Ser Cys Val Thr
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Arg Ala Glu Ala Glu Ala Val Gly Val Leu Val Lys Glu Ala Val Trp
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Ala Phe Leu Pro Asp Ala Phe Val Thr Met Thr Gly Gly Phe Arg Arg
325 330 335
Gly Lys Lys Ile Gly His Asp Val Asp Phe Leu Ile Thr Ser Pro Gly
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Ser Ala Glu Asp Glu Glu Gln Leu Leu Pro Lys Val Ile Asn Leu Trp
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Glu Lys Lys Gly Leu Leu Leu Tyr Tyr Asp Leu Val Glu Ser Thr Phe
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Glu Lys Phe Lys Leu Pro Ser Arg Gln Val Asp Thr Leu Asp His Phe
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Gln Lys Cys Phe Leu Ile Leu Lys Leu His His Gln Arg Val Asp Ser
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Ser Lys Ser Asn Gln Gln Glu Gly Lys Thr Trp Lys Ala Ile Arg Val
420 425 430
Asp Leu Val Met Cys Pro Tyr Glu Asn Arg Ala Phe Ala Leu Leu Gly
435 440 445
Trp Thr Gly Ser Arg Gln Phe Glu Arg Asp Ile Arg Arg Tyr Ala Thr
450 455 460
His Glu Arg Lys Met Met Leu Asp Asn His Ala Leu Tyr Asp Lys Thr
465 470 475 480
Lys Arg Val Phe Leu Lys Ala Glu Ser Glu Glu Glu Ile Phe Ala His
485 490 495
Leu Gly Leu Asp Tyr Ile Glu Pro Trp Glu Arg Asn Ala
500 505
Claims (8)
1.一种末端脱氧核苷酸转移酶的突变体,其特征在于:
该突变体氨基酸序列为基于SEQ ID NO:2所示的氨基酸序列的P215、F267和G372的氨基酸残基处三个氨基酸取代的氨基酸序列;其中所述三个氨基酸取代选自:P215E,F267G,G372Q;或者,P215D,F267A,G372Q;或者,P215G,F267I,G372T;
所述突变体能够在没有模板的情况下合成核酸片段;
所述突变体能够将修饰的核苷酸掺入到核酸片段中。
2.根据权利要求1所述的末端脱氧核苷酸转移酶的突变体,其中所述突变体在SEQ IDNO:2的N末端和/或C末端处包括组氨酸标签序列。
3.一种编码末端脱氧核苷酸转移酶突变体的核酸分子,所述核酸分子编码如在权利要求1-2中任一项中定义的末端脱氧核苷酸转移酶的突变体。
4.一种表达载体,所述表达载体包含权利要求3所述的核酸分子。
5.一种宿主细胞,所述宿主细胞包含权利要求3所述的核酸分子或权利要求4所述的表达载体。
6.用于产生如权利要求1-2中任一项中所定义的末端脱氧核苷酸转移酶的突变体的方法,其中根据权利要求5所述的宿主细胞在允许编码所述突变体的核酸分子表达的培养条件下被培养,并且其中所述突变体被回收。
7.如权利要求1-2中任一项中所定义的末端脱氧核苷酸转移酶的突变体用于在没有模板的情况下用3’O-修饰的核苷酸合成核酸分子的用途。
8.用于在没有模板的情况下合成核酸分子的方法,所述方法包括使核酸引物与至少一种3’O-修饰的核苷酸,和如权利要求1-2中任一项中所定义的末端脱氧核苷酸转移酶的突变体接触的步骤。
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