CN108220255A - 一种嗜热古细菌的高活性dna连接酶突变体 - Google Patents

一种嗜热古细菌的高活性dna连接酶突变体 Download PDF

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CN108220255A
CN108220255A CN201710870258.0A CN201710870258A CN108220255A CN 108220255 A CN108220255 A CN 108220255A CN 201710870258 A CN201710870258 A CN 201710870258A CN 108220255 A CN108220255 A CN 108220255A
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CN108220255B (zh
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王磊
李燕
王亮
郑春阳
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TIANJIN QIANGWEITE BIO-TECH Co Ltd
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    • C12Y605/01—Ligases forming phosphoric ester bonds (6.5) forming phosphoric ester bonds (6.5.1)
    • C12Y605/01001—DNA ligase (ATP) (6.5.1.1)

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Abstract

本发明属于分子生物学技术领域，具体提供一种来源于嗜热古细菌Geoglobus acetivorans的DNA连接酶高活性突变体，并通过将该DNA连接酶高活性突变体导入到大肠杆菌中，构建了重组表达该DNA连接酶高活性突变体的大肠杆菌工程菌。同野生型的Geoglobus acetivorans DNA连接酶相比，该DNA连接酶高活性突变体具有更高的活性，可以作为分子生物试剂，应用于分子克隆和二代测序建库技术等方面。

Description

一种嗜热古细菌的高活性DNA连接酶突变体
技术领域
本发明属于分子生物学技术领域，具体提供一种来源于嗜热古细菌Geoglobus acetivorans的DNA连接酶高活性突变体及其应用。
背景技术
DNA连接酶催化形成磷酸二酯键将两段DNA连接，在生物体内负责维护DNA的完整性，在DNA复制、重组和修复等生理活动中发挥重要的功能。根据配基的不同，DNA连接酶可以分为两大类：依赖ATP的DNA连接酶和依赖NAD+的DNA连接酶。依赖ATP的DNA连接酶主要分布于真核生物、古细菌和病毒中，依赖NAD+的DNA连接酶主要分布于细菌和部分感染真核生物的病毒中。
DNA连接酶在分子生物和生物技术领域具有广泛的应用。例如，在DNA重组构建操作中，DNA连接酶已有几十年的应用历史。近年来，来源于嗜热菌Thermus aquaticus的DNA聚合酶被应用于吉布森连接(Gibson assembly)。DNA连接酶也被应用于高通量测序建库反应和部分高通量测序反应（SOLiD测序）中。
目前被广泛使用的T4 DNA连接酶是一种依赖ATP的DNA连接酶，其对平末端的连接活性较弱，且在65度以上的温度失活。而常见的来源于嗜热菌的DNA连接酶，如Taq DNA连接酶，Pfu DNA连接酶，在高温时具有很好的活性，但是在室温条件下活性较低。
发明内容
本发明的目的是提供一种在大温度范围内（20-80度）具有较高活性的DNA连接酶突变体。本发明通过构建大肠杆菌工程菌，能够高效的重组表达所述DNA连接酶突变体，同现有市售DNA连接酶相比，该酶能够在大温度范围内保持较高活性，避免了在不同温度下操作需要更换DNA连接酶的麻烦。
本发明提供了一种来源于嗜热古细菌Geoglobus acetivorans的DNA连接酶高活性突变体，包括：
氨基酸序列为SEQ ID NO:1的酶，以及与SEQ ID NO:1序列相似性高于95%，且具有DNA连接酶。编码所述DNA连接酶高活性突变体的一种核苷酸序列为SEQ ID NO:2。
本发明将上述DNA连接酶高活性突变体导入大肠杆菌中，构建了重组表达该DNA连接酶高活性突变体的大肠杆菌工程菌。所述大肠杆菌工程菌能够高效的表达转座酶高活性突变体。
本发明还涉及上述DNA连接酶高活性突变体在分子生物领域的应用。
附图说明
图1，DNA连接酶高活性突变体在不同温度下的连接酶活性。其中泳道1为不加连接酶的底物（负对照），泳道2为DNA连接酶高活性突变体在80度下的反应产物，泳道3为DNA连接酶高活性突变体在50度下的反应产物，泳道4为DNA连接酶高活性突变体在20度下的连接产物。
具体实施方式
实施例1 DNA连接酶高活性突变体大肠杆菌工程菌的构建
在嗜热古细菌Geoglobus acetivorans的DNA连接酶中引入高活性突变D537R, R541A,R544A, R551A，根据突变后的蛋白序列，参照大肠杆菌的密码子偏好，获得了密码子优化后的编码该DNA连接酶高活性突变体基因序列，所得的序列经金唯智公司合成，并构建至pET21表达载体中，得到pET21DLMut质粒
将pET21DLMut质粒转化到大肠杆菌BL21（DE3）表达菌株中，使用LB培养基，37度培养至OD600=0.6，加入0.1mM IPTG，诱导培养6h，离心收集菌体。
实施例2 DNA连接酶高活性突变体的活性测定
用于测定DNA连接酶活性的底物是两段长度约为680bp的DNA片段（通过Hind III酶切一段长度为1366bp的DNA序列获得），将适量的该底物，加入25μL含有1x DNA连接酶高活性突变体的反应缓冲液的反应体系中（每反应体系含有50U的DNA连接酶高活性突变体），在20度，50度和80度反应30分钟，1%琼脂糖凝胶检测连接反应产物。
实施例3 DNA连接酶高活性突变体在分子生物学领域的应用
DNA片段和载体DNA的连接
1.在微量离心管中制备以下连接反应液
2. 连接反应条件：22°C连接2小时或16°C连接过夜
3. 取5-10 μl连接产物转化至100 μl感受态细胞中。
序列表
<110> 天津强微特生物科技有限公司
<120> 一种嗜热古细菌的高活性DNA连接酶突变体
<160> 2
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<210> 1
<211> 559
<212> PRT
<213> DNA连接酶高活性突变体蛋白序列(人工序列)
<400> 1
Met Leu Thr Phe Ser Glu Phe Ala Glu Leu Cys Gln Thr Val Glu Lys
1 5 10 15
Ile Ser Ser Thr Leu Glu Lys Thr Ala Arg Ile Ala Val Phe Ile Arg
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Glu Ile Glu Asp Glu Asn Asp Leu Tyr Asn Ala Val Leu Phe Leu Gln
35 40 45
Gly Arg Ile Tyr Pro Glu Trp Ser Glu Arg Asp Leu Gly Val Gly Val
50 55 60
Gly Leu Ile Tyr Glu Ala Met Arg Ile Ala Thr Gly Ile Asp Arg Lys
65 70 75 80
Thr Ile Glu Asn Leu Ile Arg Glu Lys Gly Asp Phe Gly Leu Ala Ala
85 90 95
Glu Glu Leu Val Lys Lys Lys Thr Gln Thr Leu Leu Phe Thr Glu Glu
100 105 110
Leu Thr Val Lys Lys Leu Arg Glu Ile Phe Asp Glu Ile Ser Ser Leu
115 120 125
Glu Gly Gly Gly Ser Gln Lys Lys Lys Ile Leu Leu Leu Ser Glu Leu
130 135 140
Tyr Ser Leu Cys Ser Pro Ile Glu Ala Arg Tyr Leu Thr Arg Leu Ile
145 150 155 160
Leu Arg Glu Met Arg Leu Gly Ile Gly Glu Gly Ile Ile Arg Asp Ala
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Ile Gly Lys Ala Phe Gly Ile Glu Ser Glu Ile Val Glu Arg Ala Tyr
180 185 190
Met Ile Thr Asn Asp Phe Gly Lys Val Ala Val Glu Ala Lys Asn Gly
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Gly Lys Gln Ala Leu Leu Ser Leu Lys Ile Thr Pro His Ile Pro Val
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Lys Met Met Leu Ala Gln Val Ala Glu Ser Leu Glu Glu Ala Thr Arg
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Glu Ile Arg Glu Leu Gly Val Glu Trp Lys Phe Asp Gly Ser Arg Val
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Gln Ile His Tyr Ala Asp Gly Arg Val Thr Ile Tyr Ser Arg Arg Leu
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Glu Asn Val Thr Asn Ala Leu Pro Asp Ile Val Ser Glu Ile Lys Arg
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Cys Val Lys Glu Asn Val Ile Leu Asp Gly Glu Val Ile Ala Val Arg
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Asp Gly Arg Pro Met Pro Phe Gln His Val Leu Arg Arg Phe Arg Arg
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Lys Asn Leu Glu Ser Lys Tyr Ile Pro Gly Lys Arg Gly Lys Asn Trp
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<212> DNA
<213> DNA连接酶高活性突变体基因序列(人工序列)
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atgctgacct tctctgaatt cgcggaactg tgccagaccg ttgaaaaaat ctcttctacc 60
ctggaaaaaa ccgcgcgtat cgcggttttc atccgtgaaa tcgaagacga aaacgacctg 120
tacaacgcgg ttctgttcct gcagggtcgt atctacccgg aatggtctga acgtgacctg 180
ggtgttggtg ttggtctgat ctacgaagcg atgcgtatcg cgaccggtat cgaccgtaaa 240
accatcgaaa acctgatccg tgaaaaaggt gacttcggtc tggcggcgga agaactggtt 300
aaaaaaaaaa cccagaccct gctgttcacc gaagaactga ccgttaaaaa actgcgtgaa 360
atcttcgacg aaatctcttc tctggaaggt ggtggttctc agaaaaaaaa aatcctgctg 420
ctgtctgaac tgtactctct gtgctctccg atcgaagcgc gttacctgac ccgtctgatc 480
ctgcgtgaaa tgcgtctggg tatcggtgaa ggtatcatcc gtgacgcgat cggtaaagcg 540
ttcggtatcg aatctgaaat cgttgaacgt gcgtacatga tcaccaacga cttcggtaaa 600
gttgcggttg aagcgaaaaa cggtggtaaa caggcgctgc tgtctctgaa aatcaccccg 660
cacatcccgg ttaaaatgat gctggcgcag gttgcggaat ctctggaaga agcgacccgt 720
gaaatccgtg aactgggtgt tgaatggaaa ttcgacggtt ctcgtgttca gatccactac 780
gcggacggtc gtgttaccat ctactctcgt cgtctggaaa acgttaccaa cgcgctgccg 840
gacatcgttt ctgaaatcaa acgttgcgtt aaagaaaacg ttatcctgga cggtgaagtt 900
atcgcggttc gtgacggtcg tccgatgccg ttccagcacg ttctgcgtcg tttccgtcgt 960
aaacacggtg tttctcgtat ggttgaagaa atcccgctga aagtttacct gtacgacatc 1020
ctgtacgacg acggtgaagt tatcgacctg ccgctgaaag aacgtcgtgc gcgtctgatc 1080
tctgcggttg gtgaatctga cgcggttaaa gttgcggaac agatcgttac ctctgacgtt 1140
cgtgttatcc agcaggaatt cgaccgtgcg atccaggcgg gtcacgaagg tctgatgctg 1200
aaaaacctgg aatctaaata catcccgggt aaacgtggta aaaactggct gaaactgaaa 1260
gcgaccatgg aaaccctgga cctggttgtt gttggtggtg aatggggtga aggtaaacgt 1320
tctaacctga tctcttcttt cgaactggcg tgcctggacg aatacggtgg tctgctgcgt 1380
gttggtaaag ttgcgaccgg tttcaccgac gaagacctgg aagaactgac cgaactgttc 1440
aaaccggaaa tcgaatacca ggaaggtaaa cgtatcgttt tcaacccgaa atacgttttc 1500
gaagttgcgt accaggaaat ccagaaatct ccgaaatacg aatctggtta cgcgctgcgt 1560
ttcccgcgtt tcgttcgtct gcgtgacgac aaatctgttg aagaagcgcg taccgttgaa 1620
gcggttgcgg cgctgtacga agttcagttc gcgtctaaag gtggttcttg gtcttct 1677

Claims (3)

1.一种DNA连接酶高活性突变体，其特征在于所述DNA连接酶高活性突变体的氨基酸序列为SEQ ID NO:1。
2.编码权利要求1所述DNA连接酶高活性突变体的基因，其核酸序列为SEQ ID NO:2。
3.权利要求1所述转座酶高活性突变体在分子生物学领域中的应用。
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