CN117567640A - 一种在大肠杆菌宿主细胞中高效表达与纯化重组蛋白的融合标签肽及其应用 - Google Patents
一种在大肠杆菌宿主细胞中高效表达与纯化重组蛋白的融合标签肽及其应用 Download PDFInfo
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Abstract
本发明提供了一种在大肠杆菌宿主细胞中高效表达与纯化重组蛋白的融合标签肽及其应用，属于基因工程与分子生物学领域。本发明设计的融合标签肽包含一个有利于启动基因转录水平的结构域T7tag，两个亲和纯化标签结构域6×His标签和Strep II tag标签，以及一个蛋白酶识别结构域EK位点，并融合于目的重组蛋白的N端。本发明能够有效提高重组蛋白表达水平，实现目的重组蛋白的高效表达与纯化，具有很好的普适性和应用场景。
Description
技术领域
本发明属于基因工程与分子生物学技术领域，具体涉及一种在大肠杆菌宿主细胞中高效表达与纯化重组蛋白的融合标签肽及其应用。
背景技术
大肠杆菌因其具有遗传背景清楚，技术操作和培养条件简单，大规模发酵经济等优点而备受关注，同时大肠杆菌也是目前应用最为广泛的表达系统，但是该系统也存在一定的局限性，主要包括部分重组蛋白的表达水平低、可能因折叠不正确而呈包涵体表达、蛋白不稳定易降解等，并非每一种基因都能在其中有效表达，导致目的蛋白制备纯化的难度增加。因此，融合标签的发明和应用有效解决了大肠杆菌中重组蛋白表达的相关问题。融合标签技术是20世纪末兴起的一种基因重组技术，其主要过程是利用重组DNA技术在靶蛋白编码基因的3'端或5'端融合某种标签的编码基因，通过适宜的宿主来表达重组蛋白质，表达的重组蛋白质可以通过融合的标签与包被在固相基质上的特异配基结合而进行纯化。它的应用不仅便于重组蛋白的分离纯化，而且具有促进蛋白表达，介导蛋白正确折叠、增加蛋白稳定性等作用。
目前已开发出的一系列常见的融合标签，有6×His标签、Strep标签、小分子泛素样修饰蛋白(SUMO)、谷胱甘肽转移酶(GST)、麦芽糖结合蛋白(MBP)、转录终止抗终止因子(NusA)等，但是其功能比较单一，也都具有其局限性，一般仅有助于目的蛋白亲和层析纯化或是检测鉴定，一种纯化标签仅能用于一种亲和纯化，且比较大的融合标签影响重组蛋白的功能，没有一种通用的融合标签能解决大部分蛋白的高表达和获得高纯度重组蛋白的问题。因此，提供一种普适性强、表达水平高的融合标签肽对于优化和改进基于大肠杆菌的重组蛋白基因工程与分子生物学技术体系具有重要意义。
发明内容
针对现有技术存在的不足，本发明的目的在于提供一种在大肠杆菌宿主细胞中高效表达与纯化重组蛋白的融合标签肽及其应用，通过设计融合标签肽组合序列，有效提高重组蛋白表达水平，实现目标蛋白的高效纯化。
为达到此发明目的，本发明采用以下技术方案：
本发明提供一种在大肠杆菌宿主细胞中高效表达与纯化重组蛋白的融合标签肽，包含一个有利于启动基因转录水平的结构域T7 tag，两个亲和纯化标签结构域6×His标签和Strep II tag标签，以及一个蛋白酶识别结构域的EK位点；所述融合标签肽的氨基酸序列如SEQ ID NO:1所示。
本发明设计的融合标签肽的功能结构域如图1所示，融合标签肽转录RNA序列的二级结构预测如图2所示。
优选地，所述融合标签肽融合于目的重组蛋白的N端。
本发明提供一种编码所述融合标签肽的核酸，所述核酸的序列如SEQ ID NO:2所示。
本发明提供一种重组载体，包括表达载体，所述表达载体上插入有编码融合标签肽的核酸。
优选地，所述表达载体为大肠杆菌pET24a表达载体。
本发明提供一种所述重组载体的构成方法，将所述融合标签肽克隆到表达载体pET24a的NdeI和BamHI酶切位点之间，构成重组载体。
本发明提供一种利用融合标签肽提高目的蛋白表达量的方法，包括以下步骤：构建上述重组载体，将目的蛋白密码子优化后的基因片段克隆到所述重组载体上，克隆位点优选BamHI和EcoRI，然后将带有目的蛋白基因片段的重组载体转化到表达宿主中，再进行诱导表达。
优选地，所述表达宿主为大肠杆菌BL21(DE3)pLysS菌株。
本发明提供所述融合标签肽在工业化生产蛋白质药物、重组细胞因子、重组胶原蛋白、重组蛋白抗原、重组蛋白疫苗中的应用。
相对于现有技术，本发明具有以下有益效果：
本发明设计的融合标签肽长度短，核酸序列无稳定的高级结构，不仅显著提升了重组蛋白的表达量，而且具有两种高亲和性功能域，可与特定亲和树脂或亲和配体结合，从而实现目标蛋白的高效纯化。该融合标签肽末端带有特定蛋白酶识别位点，可以被特定蛋白酶高效切除，从而获得无标签重组蛋白。与现有技术相比，本发明提供的融合标签肽普适性强，且与普通His标签相比，蛋白表达量高出4倍，蛋白纯度提高了40％，具有极大的市场前景。
附图说明
图1为融合标签肽功能结构域结构图。
图2为融合标签肽转录RNA序列二级结构预测图。
图3为B2UM07蛋白放大1L的表达情况(对照组未加本发明中的融合标签)；泳道A：实验组蛋白表达情况，泳道B：对照组蛋白表达情况。
图4为ALDH2蛋白放大1L的表达情况(对照组未加本发明中的融合标签)；泳道A：实验组蛋白表达情况，泳道B：对照组蛋白表达情况。
图5为Nb-211蛋白放大1L的表达情况(对照组未加本发明中的融合标签)；泳道A：实验组蛋白表达情况，泳道B：对照组蛋白表达情况。
具体实施方式
下面通过具体实施方式来进一步说明本发明的技术方案。本领域技术人员应该明了，所述实施例仅仅是帮助理解本发明，不应视为对本发明的具体限制。
实施例中未注明具体技术或条件者，按照本领域内的文献所描述的技术或条件，或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可通过正规渠道商购买获得的常规产品。
以下实施例中所用到的材料和试剂：
菌株：E.coli BL21(DE3)pLysS蛋白表达宿主菌购自唯地公司。
LB液体培养基、LB固体培养基，100mg/mL氨苄青霉素，本公司(泓迅生物)自行制备。
1M异丙基硫代-β-D-半乳糖苷(IPTG)：称取2.31g IPTG，用6mL超纯水溶解，定容至10mL，而后用0.22μm滤膜过滤，-20℃保存。IPTG购自国药试剂公司。0.22μm滤膜购自Minisart High Flal公司。
1M DTT：称取1.543g DTT，用6mL ddH2O溶解，定容至10mL，-20℃保存。DTT购自BBI公司。
5×SDS-PAGE上样缓冲液：量取1M Tris-HCl缓冲液(pH6.8)12.5mL、甘油25mL，称取5g SDS、0.25g溴酚蓝，用ddH2O溶解，定容至80mL，4℃冰箱保存。每次使用时，按照4:1的比例，取以上溶液2mL、1M DTT 0.5mL，充分混匀后使用，4℃冰箱保存。
5×Tris-Gly电泳缓冲液：称取15.1Tris碱、94g甘氨酸、5g SDS，用约800mLddH20搅拌溶解，而后定容至1L，室温保存。
SDS-PAGE染色液：按照无水乙醇：ddH2O：冰乙酸＝4.5：4.5：1的比例配置溶液，加入1g考马斯亮蓝R250，充分溶解，而后定容至1L，室温保存。
脱色液Ⅰ：按照无水乙醇：ddH2O：冰乙酸＝25：65：8的比例配置溶液，室温保存。
脱色液Ⅱ：按无水乙醇：ddH2O：冰乙酸＝10：75：15的比例配置溶液，室温保存。
实施例1：融合标签肽基因的合成
设计氨基酸序列如SEQ ID NO:1所示的融合标签肽，本公司采用基因合成的方法制备融合标签肽基因，融合标签肽经密码子优化后的核苷酸序列如SEQ ID NO:2所示。
融合标签肽的氨基酸序列(SEQ ID NO:1)：
MASMTGGQQMGRGSHHHHHHWSHPQFEKVGTGSNDDDDK
融合标签肽的核苷酸序列(SEQ ID NO:2)：
atggcgagcatgaccggcggccagcagatgggccgtggttctcatcatcatcaccatcattggagccacccgcag tttgaaaaagtgggcaccggcagcaacgatgatgatgataaa
实施例2：以B2UM07作为目的重组蛋白基因的实验组表达载体及对照组表达载体的构建
1、实验组表达载体的构建
B2UM07的核苷酸序列(SEQ ID NO:4)：
atggagaagaacgcgccgtttagcgtgatgaacatgcatagctttcgctggattcgcctgaccgcgtttagcgcgctggcagcagcagcaattacttcttgcgcgagcgcagcgaccgattttaaccaggtgggcaaacagatgagcctgctgctgcagaactttcatttcagccgcaaagagtttagcgatgaactgagcaccaaatttctggaaacctatctgcgcaaagtggacccgaacaaaatcttctttacccagcaggatgtggatgcgctgaagcgcaaatacggcaaagagctggatgattatctgatgagcggccagatgatggatgcggcgcaggcgatgcatgcgctgtatagacagcgcgcaatgcagcgcattagctatgcgcgcgatctgctgaaaaagggcggctttacctttgataaagataagagcattgaacgcagccgccgcaaaaccgcggcgtggccgaaagatgaagcagaaatgcagcaggtgtggaaagatatggtggaagaacagctgctgagcgagattctgcgccgcgaaaccgtggcgcgcttagcgaaagaacagaacaaaccagatccgctggcgaacgaaaaaccggcggaggaaaaactgctgatgcgctatgaacgcattcagcgcaacattcaggaaaccgatctggaagatgtggcggaaaccctgctgagcgcggtggcgctgacttatgatccgcataccgattatatgggcgcgcgccaggtggatcgctttaaaattagcatgggcaccgaactgaccggcattggcgcgctgctgggttctgaagatgatggcagcaccaaaattaccggcattgtggtgggcggcccggcggataaaagcggcgaactgaaactgaacgatcgcattgtggcgattgatagcgacaacagcggcgaaatggtggatattctgtttatgaaactggacaaggtggtggatatgattcgcggcgcggaaaacacccagatgcgcctgaaagtggaaccggcagatgcgccgggccaagcaaaaattatcaccctgacccgcagcaaagtgccgctgaaagatgaactggcgaaaggcgaaattattgaactgaccggcgcgccggaaggccgcaaccgcattggcgtgttatctttaccaagcttttatgcggatatggaaggcggcgatcgccgctgcgcgaaagacgtgaagaaaattctggaacgcatgaacaaagaaaacgtggatggcctggtgattgatctgcgcagcaacggcggcggcagcttagaagaagtgcgcctgatgactggcttctttaccggcaacggcccggtggtgcagattaaagatacccgcggcaacgtggatattaaaagcgcgcataaccgccagaaactgtttaacggcccgattgtggtgctgattaacaaactgagcgcgagcgcgagcgaaattctggcggcggcgttacaggattatggccgtgcggtgattgtgggcgatgaaagcacctttggcaaaggcagcgtgcagcagccggtggatattggccagtatctgccgtttttcgcggcgcgcgatcgcgcgggcttgttgaaagtgaccactcagaaattttatcgcgtggcgggcggcagcacccagctgaaaggtgtggaaagcgatattcagctgccgaccgcgaccgcggcgtttgaactgggcgaagatattctggattatgcgatgccgtatgatcagattaccccgtgcaccaactataaaaaggatagcagcattgcggcgatgctgccggtgctgaaagatgcgagcgcgaaacgcgtggaaaaagatcgcgatctgcagattgcgcgcgaagatattgcgatgatgaaacagcgcattaaagataacaagctgagcctgaacaaaaagattcgcgaacaggaaaacagcgcgctggaagaacgccgcaaaagcattaacaaagaacgcaaaattcgctttgcggaaatggcgcgcgaagatgcgaccaaatataagatttatcgcctgaccctggatgatgtgaacgcgaaagaactgccgctggcggacccggaaaaagataacgaacagtttatgcatctggcggaagatccgaccgcggaactggatgatagcccggaatatccgagcggcttggacccggaactgcgcgagggcattaacattgtgcaggatatgctgaaactggaaagcagcggcaaataa
将实施例1中合成的融合标签肽基因克隆到表达载体pET24a的NdeI和BamHI酶切位点之间，构成重组载体，重组载体的核苷酸序列如SEQ ID NO:3所示；将B2UM07密码子优化后的基因片段(SEQ ID NO:4)，通过BamHI和EcoRI酶切位点克隆到上述重组载体中构成实验组表达载体。
2、对照组表达载体的构建
将B2UM07密码子优化后的基因片段(SEQ ID NO:4)，克隆到表达载体pET24a的NdeI和XhoI酶切位点之间，去掉目的基因的终止密码子，使用载体上的终止密码子终止翻译，形成对照组表达载体。
实施例3：以ALDH2作为目的重组蛋白基因的实验组表达载体及对照组表达载体的构建
ALDH2的核苷酸序列(SEQ ID NO:5)：
atgctgcgcgcggcggcgcgctttggcccgcgtttaggtcgtcgtttattatctgcggcggcgactcaagcggttccggcaccaaaccaacaaccggaagtgttttgcaaccagatttttatcaacaacgaatggcatgatgcggtgagccgcaaaacctttccgaccgtgaacccgagcaccggtgaagtgatttgccaggtggcggaaggcgataaagaagatgtggataaagcggtgaaagcggcgcgcgcggcgtttcaattaggttctccatggcgtcgcatggatgcgagccatcgcggccgcttactgaaccgtttagcggatctgattgaacgcgatcgcacctatctggcggcgctggaaaccctggataacggcaaaccgtatgtgattagctatctggtggatctggatatggtgctgaaatgcctgcgctattatgcgggctgggcggataaataccacggcaaaaccattccgattgatggcgatttctttagctatacccgccatgaaccggtgggcgtgtgcggccagattattccgtggaactttccgctgctgatgcaggcgtggaaactgggcccggcgttagcgactggtaacgtggtggtgatgaaagtggcggaacagaccccgctgaccgcgctgtatgtggcgaacctgattaaagaagcgggctttccgccgggcgtggtgaacattgtgccgggttttggtccaactgcaggtgcggcgattgcgagccatgaagatgtggataaagtggcgtttaccggcagcaccgaaattggccgcgtgattcaggtggcagcaggcagtagcaacctgaaacgcgtgaccctggagctgggtggcaaaagcccgaacattattatgagcgatgcggatatggattgggcggtggaacaggcgcattttgcgctgtttttcaaccagggccagtgctgctgcgcgggtagccgtacttttgtgcaggaagacatttatgatgaatttgtggaacgcagcgtggcgcgcgcgaaaagccgcgttgttggcaatccatttgatagcaaaaccgaacagggcccgcaggtggacgagacccagtttaaaaagatcctgggctatattaacaccggcaaacaggaaggcgcgaaactgctgtgcggcggcggtattgcagcagatcgtggctattttattcagccgaccgtgtttggcgatgtgcaggatggcatgaccattgcgaaggaggagatctttggcccggtgatgcagattctgaaatttaaaaccattgaagaagtggtgggccgcgcgaacaacagcacttatggcctggcggcagcagtgttcaccaaagatctggataaagcgaactatctgagccaggcgctgcaggcgggcaccgtgtgggtgaattgctatgatgtgtttggcgcgcagagcccgtttggcggctataaaatgagcggcagcggtcgtgaactgggcgaatatggcctgcaggcgtataccggtgtgaaaaccgtgaccgtgaaagtgccgcagaaaaacagctaa
以ALDH2密码子优化后的基因片段(SEQ ID NO:5)作为目的重组蛋白基因构建实验组表达载体和对照组表达载体，构建方法参照实施例2。
实施例4：以Nb-211作为目的重组蛋白基因的实验组表达载体及对照组表达载体的构建
Nb-211的核苷酸序列(SEQ ID NO:6)：
tgcggcggcgaagaaccgcagaacgcgaaacgcaaactgattgtggcgaccgatgcgaccctgccgccgatgagctttctgaacgatcagaaccgcttagcgggctttgaagtggatctgattggcgcggtggcgcgcgaagcgggttttgaatatgacctgattaacgtggaatggaacggcctgtttggcggcctgattaccaagaagtatgatctggtgattagcagcgtgaccattctggaagaacgcaaagaacgcatggcgtttagcgtgccgtatctgcagagcggcctgagcctggtggttcgtcgcgatactgaaggcgtgaccagcttagaagatgtgcaggcgcaggatggcgttgttggtgcgcaacgtgcaaccaccgcgttcttttatctggaagattatccggaactgaacaaacaggcgtatgaactgtatggccatgcgatccaggatctgattaaaggcgaaattaccgcggtgctgggcgaaagcaccggcaccctgtattataaaaacaacgatgcggcggtgtttcgcgaaattaagatggtgggcgatattctgaccgaagaacattatggcattgtggcgcgcaaaggcgaaaccgaactgctgcagcgcgtgaacgatgcgctgaagaaactgctggatgatggcaccgtgcagcgcctgcatgaaaaatgggaactgggccaggcggcgatggtgccgaaaaccgtggcgtctggtaaagaaaactaa
以Nb-211密码子优化后的基因片段(SEQ ID NO:6)作为目的重组蛋白基因构建实验组表达载体和对照组表达载体，构建方法参照实施例2。
实施例5：质粒的转化和诱导表达
1、将实施例2-4中的实验组表达载体与对照组表达载体质粒各取1μL分别加入100μLBL21(DE3)pLysS感受态细菌中，置于冰上20min；
2、42℃热激90sec，迅速置冰中3min；加入500μL LB培养液；
3、37℃，220rpm振摇培养1h，取200μL菌液涂布于含50μg/mL Kan的LB平板，37℃倒置培养过夜；
4、次日早晨每组各挑取2个BL21(DE3)pLysS的单克隆分别接种于含50μg/mL Kan的4mL LB培养液的试管中，37℃、220rpm振摇培养至OD约1.5，取500μL菌液保存菌种，其余全部接入1L发酵培养基；
5、上一步所接种的1L发酵培养基，37℃、220rpm振摇培养至OD约0.5加入诱导剂IPTG至终浓度为1mM，220rpm，16℃过夜培养；
6、6000rpm，5min离心去上清收集发酵菌体，破碎纯化；
7、捕获纯化选用镍柱亲和层析，20mM Tris，500mM NaCl(pH＝7.4)作为平衡缓冲液，在咪唑洗脱液中均有目标蛋白；
8、精细纯化选用Strep II tag亲和填料进行亲和层析，20mM Tris，500mM NaCl(pH＝7.4)作为平衡缓冲液，硫化生物素洗脱目的蛋白；
9、SDS-PAGE分析重组蛋白纯化产物，实施例2-4本身的蛋白大小分别约为88.22kDa，60.67kDa，32.07kDa，与预期一致，与仅带有His标签的重组蛋白相比，带有本发明融合标签的目的重组蛋白表达水平及所得产物的纯度提高显著，SDS-PAGE的结果见图3、图4、图5。
实施例6：融合标签肽的普适性实验
为了证明本发明设计的融合标签肽的普遍适应性，分别选择B2UM07、ALDH2、Nb-211作为待表达基因进行对比实验，这3个基因单独表达时无表达，和其他标签结合表达量低，而应用本发明实施例1中的N端融合标签肽后，实验结果如图3-图5所示，具体如下：
B2UM07蛋白表达情况：应用本发明融合标签肽的B2UM07蛋白相比普通His标签的表达量高出约4倍，蛋白纯度提高了约40％。
ALDH2蛋白表达情况：应用本发明融合标签肽的ALDH2蛋白相比普通His标签的表达量高出约2倍，蛋白纯度提高了约10％。
Nb-211蛋白表达情况：应用本发明融合标签肽的Nb-211蛋白相比普通His标签的表达量高出约2.4倍，蛋白纯度提高了约20％。
申请人声明，以上所述仅为本发明的具体实施方式，但本发明的保护范围并不局限于此，所属技术领域的技术人员应该明了，任何属于本技术领域的技术人员在本发明揭露的技术范围内，可轻易想到的变化或替换，均落在本发明的保护范围和公开范围之内。
Claims (9)
1.一种在大肠杆菌宿主细胞中高效表达与纯化重组蛋白的融合标签肽，其特征在于，包含一个有利于启动基因转录水平的结构域T7 tag，两个亲和纯化标签结构域6×His标签和Strep II tag标签，以及一个蛋白酶识别结构域的EK位点；所述融合标签肽的氨基酸序列如SEQ ID NO:1所示。
2.根据权利要求1所述的融合标签肽，其特征在于，所述融合标签肽融合于目的重组蛋白的N端。
3.一种编码如权利要求1所述融合标签肽的核酸，其特征在于，所述核酸的序列如SEQID NO:2所示。
4.一种重组载体，其特征在于，包括表达载体，所述表达载体上插入有权利要求3所述编码融合标签肽的核酸。
5.根据权利要求4所述的重组载体，其特征在于，所述表达载体为大肠杆菌pET24a表达载体。
6.一种如权利要求4所述重组载体的构成方法，其特征在于，将所述融合标签肽克隆到表达载体pET24a的NdeI和BamHI酶切位点之间，构成重组载体。
7.一种利用融合标签肽提高目的蛋白表达量的方法，其特征在于，包括以下步骤：构建如权利要求4所述的重组载体，将目的蛋白的基因片段克隆到所述重组载体上，然后将带有目的蛋白基因片段的重组载体转化到表达宿主中，再进行诱导表达。
8.根据权利要求7所述的方法，其特征在于，所述表达宿主为大肠杆菌BL21(DE3)pLysS菌株。
9.权利要求1或2所述的融合标签肽在工业化生产蛋白质药物、重组细胞因子、重组胶原蛋白、重组蛋白抗原、重组蛋白疫苗中的应用。
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| CN106543272A (zh) * | 2015-09-18 | 2017-03-29 | 江苏众红生物工程创药研究院有限公司 | 多功能标签融合蛋白及其表达方法和应用 |
| CN111378047A (zh) * | 2018-12-28 | 2020-07-07 | 复旦大学 | 一种提高蛋白表达的融合标签蛋白及其应用 |
| US20210079363A1 (en) * | 2017-01-30 | 2021-03-18 | Obschestvo s ogranichennoy otvetstvennostyu "PLANTA" | Novel luciferases and methods for using same |
| CN115992159A (zh) * | 2022-07-20 | 2023-04-21 | 上海交通大学 | 一种获得目标蛋白的方法、融合蛋白、载体及应用 |
| CN116462771A (zh) * | 2023-04-26 | 2023-07-21 | 华东理工大学 | 一种利用标签制备多肽的方法 |
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| CN106543272A (zh) * | 2015-09-18 | 2017-03-29 | 江苏众红生物工程创药研究院有限公司 | 多功能标签融合蛋白及其表达方法和应用 |
| US20210079363A1 (en) * | 2017-01-30 | 2021-03-18 | Obschestvo s ogranichennoy otvetstvennostyu "PLANTA" | Novel luciferases and methods for using same |
| CN111378047A (zh) * | 2018-12-28 | 2020-07-07 | 复旦大学 | 一种提高蛋白表达的融合标签蛋白及其应用 |
| CN115992159A (zh) * | 2022-07-20 | 2023-04-21 | 上海交通大学 | 一种获得目标蛋白的方法、融合蛋白、载体及应用 |
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