CN114107252A - CL7蛋白质、高活性重组TET酶CL7-NgTET1、其原核表达载体及应用 - Google Patents
CL7蛋白质、高活性重组TET酶CL7-NgTET1、其原核表达载体及应用 Download PDFInfo
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- CN114107252A CN114107252A CN202111476804.5A CN202111476804A CN114107252A CN 114107252 A CN114107252 A CN 114107252A CN 202111476804 A CN202111476804 A CN 202111476804A CN 114107252 A CN114107252 A CN 114107252A
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Abstract
本发明提供一种屏蔽核酸酶活性的CL7蛋白质,其氨基酸序列如SEQ ID NO.1所示。还公开了一种高活性重组TET酶CL7‑NgTET1,其氨基酸序列如SEQ ID NO:4所示,及其原核表达载体,其结构为:pET28A‑N6H‑MBP‑CL7‑NgTET1,还公开了其在酶转化法检测DNA甲基化中的应用。本发明在NgTET1基础上进行了设计和改造,通过融合CL7 domain获得了CL7‑NgTET1重组融合蛋白。该重组融合蛋白能够显著提高TET蛋白对5mC的氧化能力,提高了该酶的体外生物活力。通过在载体N端引入MBP融合蛋白,可在原核***中表达,从而极大提高可溶蛋白的产量,从而同时解决了TET酶产量低和酶活低的问题,也极大缩短了生产周期及降低成本。
Description
技术领域
本发明专利涉及一种CL7蛋白质、高活性重组TET酶CL7-NgTET1、其原核表达载体及应用,属于生物技术领域。
背景技术
DNA羟甲基化酶,Ten Eleven Translocase(以下简称TET)是真核生物中普遍存在的一种α-酮戊二酸和Fe2+依赖的双加氧酶,在生物进化过程中具有高度的保守性。TET酶是DNA去甲基化过程的关键蛋白,可以将5mC通过三步氧化反应(5mC-5hmC-5fC-5caC)转变成5caC。现有主要的TET酶包括:鼠源mTET1/2、人源hTET1/2、NgTET1等及其系列突变、截短或融合。
其中具有较高功能活性的重组鼠源和人源TET酶主要通过人293系列细胞获得,却难以在原核细胞中表达,因此也极大限制了该酶的产量问题和极大提高了生产成本及产出周期。而重组NgTET1以往观点认为其在体外反应时,对5mC的氧化能力相对较低,因而在体外单酶生产及应用方面该酶的深度开发程度相对较低。
Yeasen公司在获得重组NgTET1在大肠杆菌中高表达后,自研的5mC酶法转化试剂盒检测到其较好的甲基化胞嘧啶的转化效率,但仍相对低于行业顶尖标准。对于严格要求甲基化检出的肿瘤早筛等应用市场,低转化意味着低效率和低准确性,原活性的NgTET1应用前景有限。因此,基于行业痛点和公司实验室基础,本发明对NgTET1引入了ColE7的改造。
ColE7(以下简称CL7)是来源于大肠杆菌素的核酸酶domain蛋白,其良好的与核酸结合能力,近期被用来进行突变改造屏蔽其核酸酶活性后与人造重组蛋白进行融合,从而提高蛋白某方面的生物活性。例如,Y Wang等对Taq DNA polymerase的改造,提高了其DNA扩增的能力。也有通过其与Im7特异结合的机理来生产层析用填料,用作一步高效的亲和蛋白纯化(Dmitry G.Vassylyev,2017 Efficient,ultra-high affinity chromatographyin a one-step purification of complex proteins)。
发明内容
本发明的目的是提供一种CL7蛋白质,以及其在提高TET酶NgTET1的活性中的应用。
本发明采用的技术方案为:一种CL7蛋白质,其特征在于:其氨基酸序列如SEQ IDNO.1所示。
本发明还公开了一种高活性重组TET酶CL7-NgTET1,其特征在于:其氨基酸序列如SEQ ID NO:4所示,野生型NgTET1的氨基酸序列如SEQ ID NO:2所示。
本发明还公开了上述的高活性重组TET酶CL7-NgTET1的原核表达载体,其特征在于其结构为:pET28A-N6H-MBP-CL7-NgTET1,其中N6H为N端His标签,MBP编码SEQ ID NO:3所示的MBP融合蛋白。
优选的,N6H与MBP之间通过linker连接,所述linker的具体序列为AGCAGCGGCTTTGGTGCCGCGCGGCAGCCAT。
优选的,MBP与CL7-NgTET1之间设有TEV蛋白酶位点,其核酸序列为ATCTGTATTTTCAGGGT。
本发明还公开了上述的高活性重组TET酶CL7-NgTET1在酶转化法检测DNA甲基化中的应用。
优选的,其步骤包括:
(1)在待测样本中加入TET酶氧化缓冲液和高活性重组TET酶CL7-NgTET1;
(2)加入蛋白酶K终止反应,回收DNA;
(3)DNA甲基化检测分析。
本发明的有益效果为:
本发明在NgTET1基础上进行了设计和改造,通过融合CL7 domain获得了CL7-NgTET1重组融合蛋白。该重组融合蛋白能够显著提高TET蛋白对5mC的氧化能力,提高了该酶的体外生物活力。通过在载体N端引入MBP融合蛋白,可在原核***中表达,从而极大提高可溶蛋白的产量,从而同时解决了TET酶产量低和酶活低的问题,也极大缩短了生产周期及降低成本。本发明的高活性的重组TET酶可以高效生产,极大降低了生产成本及推动了TET酶应用的产业化进程。特别对当下的肿瘤早筛领域的新兴发展,具有极大的推动和促进作用。
附图说明
图1重组CL7-NgTET1载体构建示意图。
图2重组高活性CL7-NgTET1在Rossetta(DE3)表达的SDS-PAGE及酶切验证。
图3重组CL7-NgTET1原核与mTET1CD真核系列表达水平的比较。
图4重组CL7-NgTET1与NgTET1的性能比较。
图5重组CL7-NgTET1、NgTET1与mTET1CD的酶活检测结果。
图6重组CL7-NgTET1、NgTET1与mTET1CD氧化动力学测试结果。
具体实施方式
实施例1:重组CL7-NgTET1载体构建
(1)基因合成:按SEQ ID NO.1,SEQ ID NO.2,SEQ ID NO.3蛋白序列为模板进行合成;
(2)引物设计:按以上片段两端同pET28a载体切入点两端各20bp左右位置为同源臂设计扩增组装引物。引物序列如下:
M-Forward:5’-GTGCCGCGCGGCAGCCATATGAAAATCGAAGAAGGT-3’,
M-Reverse:5’-CGGTTCGTTAGATTTGGAACCCTGAAAATACAGATT-3’,
CN-Forward:5’-AATCTGTATTTTCAGGGTTCCAAATCTAACGAACCG-3’
CN-Reverse:5’-TGCTCGAGTGCGGCCGCTTATTTGGTTTCTTTATGA-3’
V-Forward:5’-ATAAGCGGCCGCACTCGAGCA-3’
V-Reverse:5’-ACCTTCTTCGATTTTCATATGG-3’
(3)基因扩增和胶回收:采用KOD高保真DNA聚合酶扩增体系,加入上述引物和模板,组成50uL的反应体系,用程序98℃,2min-94℃,10s-60℃,20s-68℃,3min-68℃,10min,4℃,30min在PCR仪器中循环2-4步骤30次进行扩增。琼脂糖凝胶用Omega胶回收试剂盒进行片段回收,并用nanodrop测定浓度。其中M-Forward/Reverse、CN-Forward/Reverse、V-Forward/Reverse三对引物分别以MBP、CL7-NgTET1基因、pET28a空载质粒为模板,扩增后产物分别命名为MBP片段、CL7-NgTET1片段、pET28A重组载体。
(4)片段载体组装:使用同源重组试剂盒(Gibson Assembly)对PCR回收的MBP片段、CL7-NgTET1片段及pET28A载体进行组装,反应体系如下:
组分 | 用量 |
MBP片段 | 0.2pmols |
CL7-NgTET1片段 | 0.3pmols |
pET28A重组载体 | 100ng |
2×同源重组mix | 10μL |
补ddH2O至 | 20μL |
如图1所示,完成pET28A-N6H-MBP-CL7-NgTET1重组基因组结构的构建,并保留载体N端His标签用于纯化。
实施例2:重组高活性CL7-NgTET1在Rossetta(DE3)表达的SDS-PAGE及酶切验证
具体实施步骤如下:
(1)转化:从的超低温冰箱中取出Rossetta(DE3)感受态细胞置于冰上融化后加入连接产物在冰上放置水浴中于42℃下热激再置于冰上放置加入室温无抗LB液体培养基,放置在摇床中于37℃以180r/min的转速震荡培养60min;将获得的菌液混匀后涂至浓度为的卡那霉素抗性LB平板上,平板倒置,于37℃下培养直至待单克隆菌斑形成;
(2)诱导:挑取四个单克隆,分别接种至此含有的卡那霉素的LB试管中,在摇床中于37℃下以250r/min的转速震荡培养,当OD600达到时,向其中的三个试管中分别加入0.1~1.0mM异丙基-β-D-硫代半乳糖昔,一支试管于16℃下培养 一支试管于25℃下培养一支试管于37℃下培养未加入异丙基-β-D-硫代半乳糖昔的试管作为阴性参照,采用聚丙烯酰胺凝胶电泳和蛋白质印迹法检测蛋白质的表达;
培养基与裂解缓冲液的体积比为5:1;裂解缓冲液为50mM Tris-HCl,150mMNaCl,5%glycerol,pH 8.0;
(5)上清样品:取剩余的裂解液,于的转速下离心取上清液;将5×的蛋白上样缓冲液混合到上清液中作为上清样品;蛋白上样缓冲液与上清液的体积比为1:4;采用聚丙烯酰胺凝胶电泳检测全菌、及裂解上清,得到MBP-CL7-NgTET1。
(6)上清样品加入TEV蛋白酶,室温2~4h,采用聚丙烯酰胺凝胶电泳检测,得到CL7-NgTET1。
实验结果如图2所示,MBP-CL7-NgTET1在原核Rossetta(DE3)体系中有高可溶性表达,16℃条件下最优。
实施例3:重组CL7-NgTET1原核与mTET1CD真核系列表达水平的比较。
在本实施例中,通过SDS-PAGE及BCA等法分析CL7-NgTET1在大肠Rossetta菌株中的表达水平,及mTET在293细胞中的表达水平,进而对比。
重组CL7-NgTET1在原核中表达操作同上述实施例2。取四组样品跑胶,一组为空白对照,另外三组为16℃诱导条件下的重复,分上清和沉淀进行SDS-PAGE。
GST-mTET在293细胞中的表达操作如下:
(1)质粒和细胞准备:将mTET1CD的N端采用GST融合,并构建至pEE12.4表达载体,形成pEE12.4-GST-mTET1CD质粒,用大肠杆菌DH5α扩增pEE12.4-GST-mTET1CD质粒,抽提100ug以上备用;用OPM-293CD05 Medium悬浮培养培养100ml expi293F,细胞计数至浓度为4X 106cells/ml传至10ml/瓶(4瓶,其中1瓶作为空白对照组,另外3瓶用于转染组重复)。
(2)转染:按照培养体积(ml)与质粒的量(ug)比例为1:1转染。取转染试剂PEI30uL和质粒10ug分别与0.5ml培养基混匀;再将二者混合液斡旋后静置20min;滴加入细胞瓶培养,继续培养5h(4-6h)补加0.5ml 4%的OPM-293ProFeed无蛋白补料悬浮培养。
(3)收集:培养48h后3000g,4℃,离心30min分别收集4瓶细胞。弃上清,沉淀用1mlLysis buffer(50mM Tris-Cl pH=7.5,500mM NaCl,1X蛋白酶抑制剂,1mM PMSF,1%Triton X-100)重悬。
实验结果如图3所示,CL7-NgTET1在原核中的表达要显著高于mTET1CD在293细胞中的表达水平。
实施例4:重组CL7-NgTET1与NgTET1的性能比较。
在本实施例中,利用MAPS-qPCR和MAPS-seq来验证重组CL7-NgTET1与NgTET1在DNA甲基化检测应用中的效率。
1)在待测样本(Yeasen的DNA甲基化标准品5mC oligo)中加入TET酶氧化缓冲液和重组CL7-NgTET1或NgTET11酶:
37℃反应10-60min。
反应结束后,加入0.3-3μL 10mg/mL蛋白酶K,50℃反应10-30min。使用磁珠法回收处理后的DNA。
2)过氧钨酸盐处理:
组分 | 用量 |
上述回收的DNA | 13μL |
10×PBS缓冲液 | 2μL |
200mM过氧钨酸钾 | 5μL |
补ddH2O至 | 20μL |
70℃处理3h。反应结束后,使用磁珠法回收处理后的DNA。
3)qPCR验证TET-MAPS检测m5C修饰的效率:
反应用到的引物序列为:PCR阻断探针5’-AT/LNA-C/GAAATTAATACGACT/ddC/-3’;C-引物5’-CACTGCTTACTGGCTTATC-3’;T-引物5’-CACTGCTTACTGGCTTATT-3’;引物R5’-TGGACGTACCGTGACGATG-3’;反应程序:
4)DNA NGS验证TET-MAPS检测m5C修饰的效率。
回收的DNA使用翊圣生物的HieffUltima DNA Library Prep Kit forIllumina进行DNA文库构建,构建好的文库使用illumina的NovaSeq 6000进行测序。获得的数据分析5mC转化成T的效率
结果见图4,相较于NgTET1,CL7-NgTET1具有更高的MAPS-seq检测DNA甲基化的效率。
实施例5:重组CL7-NgTET1、NgTET1与mTET1CD的酶活检测。
在本实施例中,使用Epigentek的Epigenase 5mC-羟化酶TET活性/抑制分析试剂盒(荧光法)按照说明书的流程来测定重组CL7-NgTET1与NgTET1将5mC转化成5hmC的酶比活性。
结果见图5,CL7-NgTET1具有最高的酶比活性,比活依次顺序为CL7-NgTET1>mTET1CD>NgTET1。
实施例6:CL7-NgTET1、NgTET1与mTET1CD的氧化动力学测试。
在本实施例中,测定了酶对5mC的氧化动力学曲线,反应体系如下:
组分 | 用量 |
5mC oligo | 1-100ng |
10×TET buffer | 3μL |
3mM Fe(NH4)2(SO4)2 | 1-10μL |
10μM TET酶 | 2-10μL |
补ddH2O至 | 30μL |
37℃反应10-60min。
反应结束后,加入0.3-3μL 10mg/mL蛋白酶K,50℃反应10-30min。使用磁珠法或者QIAquick Nucleotide Removal Kit(Qiagen)回收DNA进行LC-MS/MS分析。5mC、5hmC、5fC和5caC含量占比分析流程见Hideharu Hashimoto et al(Nature,2013)。
实验结果如图6所示,优化体系中CL7-NgTET1的氧化产物中5caC的占比最高。
综上,本发明提供了一种高活性重组TET酶的高效实现方法,CL7-NgTET1融合MBPtag在原核中高度表达,且CL7-NgTET1可以在体外高效地将5mC高效特异地转化成5hmC,显著提高DNA甲基化检测的效率和灵敏度,解决了TET酶产量低或酶活低的问题,将极大推进该酶在肿瘤早筛市场的迅速产业化应用。
序列表
<110> 翌圣生物科技(上海)股份有限公司
<120> CL7蛋白质、高活性重组TET酶CL7-NgTET1、其原核表达载体及应用
<141> 2021-11-22
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Claims (8)
1.一种CL7蛋白质,其特征在于:其氨基酸序列如SEQ ID NO. 1所示。
2.权利要求1所述的CL7蛋白质在提高TET酶NgTET1的活性中的应用。
3.一种高活性重组TET酶CL7-NgTET1,其特征在于:其氨基酸序列如SEQ ID NO:4所示。
4.权利要求3所述的高活性重组TET酶CL7-NgTET1的原核表达载体,其特征在于其结构为:pET28A-N6H-MBP-CL7-NgTET1,其中N6H为pET28A 质粒自带的N端His标签,MBP编码SEQID NO:3所示的MBP融合蛋白。
5.根据权利要求4所述的高活性重组TET酶CL7-NgTET1的原核表达载体,其特征在于:N6H与MBP之间通过linker连接,所述linker的序列具体为AGCAGCGGCTTTGGTGCCGCGCGGCAGCCAT。
6.根据权利要求4所述的高活性重组TET酶CL7-NgTET1的原核表达载体,其特征在于:MBP与CL7-NgTET1之间设有TEV蛋白酶位点,其核酸序列为ATCTGTATTTTCAGGGT 。
7.权利要求3所述的高活性重组TET酶CL7-NgTET1在酶转化法检测DNA甲基化中的应用。
8.根据权利要求7所述的应用,其特征在于其步骤包括:
(1)在待测样本中加入TET酶氧化缓冲液和高活性重组TET酶CL7-NgTET1;
(2)加入蛋白酶K终止反应,回收DNA;
(3)DNA甲基化检测分析。
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