CN113832127A - 一种限制性内切酶BamHⅠ的突变体及其应用 - Google Patents
一种限制性内切酶BamHⅠ的突变体及其应用 Download PDFInfo
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- CN113832127A CN113832127A CN202111253376.XA CN202111253376A CN113832127A CN 113832127 A CN113832127 A CN 113832127A CN 202111253376 A CN202111253376 A CN 202111253376A CN 113832127 A CN113832127 A CN 113832127A
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Abstract
本发明公开了一种限制性内切酶BamHⅠ的突变体及其应用,涉及内切酶突变体制备技术领域。本发明提供了一种限制性内切酶BamHⅠ的突变体,与野生型限制性内切酶BamHⅠ相比,在第197位的氨基酸发生了突变。由甘氨酸突变为天冬氨酸。突变后的突变体丧失了切割特定核苷酸序列的功能,其在大肠杆菌中可直接表达,表达的重组蛋白经亲和层析纯化后可得大量的目标蛋白。
Description
技术领域
本发明涉及内切酶突变体制备技术领域,具体而言,涉及一种限制性内切酶BamHⅠ的突变体及其应用。
背景技术
酶,又称为生物催化剂,是活细胞内产生的具有高度专一性和催化效率的蛋白质(核酶Ribozyme除外)。分子生物学研究过程中发现的酶,很多都被开发成工具来使用。这些工具酶的使用,反过来又可以促进分子生物学研究的进展。随着现代生物技术产业的兴起,工具酶在基因工程产品开发中也得到了广泛应用。
分子生物学工具酶是体外进行核酸合成、切割、修饰和连接等反应所需要的酶的统称。根据功能的不同,可以将工具酶大致分为聚合酶、限制性内切酶、修饰酶和连接酶四大类。
Ⅱ型限制性内切酶素有“分子手术刀”的美誉,能够精确切割DNA分子,是一类重要的分子生物学工具酶,在分子克隆、基因分型、遗传突变研究和测序等方面应用广泛。
早期的限制性内切酶直接从产酶的微生物中提取,经纯化得到天然产品。因为天然的酶蛋白在细胞中的含量很低,因而纯化难度大,产量有限,导致产品价格高昂。现代基因工程技术的发展使得克隆并表达限制性内切酶成为可能。而相对于天然提取,重组表达具有诸多优势,如:可利用质粒的多拷贝性或将目标基因置于强启动子下表达以提高酶蛋白的表达水平;可对菌种(如大肠杆菌)进行高密度培养,以提高限制性内切酶的产量;在构建表达载体时加入纯化标签(如His Tag)以简化分离纯化步骤等。因为限制性内切酶的表达一般不需要翻译后修饰,可以选择简单的大肠杆菌重组表达系统。
目前,限制性内切酶的重组表达仍存在表达量少、重组表达产物获取困难等难题。这是因为大多数重组表达系统或多或少存在泄露表达,而限制性内切酶表达后会切割宿主细胞DNA,从而导致菌体无法存活。目前的解决方案是,先将这种酶的修饰酶基因导入菌体并预先表达,或者通过这种酶的限制-修饰调控序列来精确调控两种酶的表达顺序。
鉴于此,特提出本发明。
发明内容
本发明的目的在于提供一种限制性内切酶BamH Ⅰ的突变体及其应用以解决上述技术问题。
表达酶的基因可以从产酶的原始菌株中调取,也可以人工合成。我们在合成限制性内切酶BamH Ⅰ的基因时,发现将编码限制性内切酶BamH Ⅰ的基因的第590位由G突变为A后,会导致翻译后的氨基酸序列第197位由甘氨酸(Glycine,G)突变为天冬氨酸(Asparticacid,D)。这段序列引入表达载体后,可以直接转化大肠杆菌并进行表达,表达产物经简单的纯化后可得到大量目标蛋白。突变体蛋白经检测失去了切割特定DNA序列的功能。因其易于大量制备,后续可用于制作蛋白Marker产品中的特定分子量条带。
本发明是这样实现的:
本发明提供了一种限制性内切酶BamH Ⅰ的突变体,其具有如SEQ ID NO.1所示的氨基酸序列。
本发明还提供了一种核酸分子,其编码上述的限制性内切酶BamH Ⅰ的突变体。
在本发明应用较佳的实施方式中,上述核酸分子具有SEQ ID NO.2所示的核苷酸序列。
本发明还提供了一种载体,其含有上述的核酸分子。
本发明还提供了一种重组菌或重组细胞,其含有上述的核酸分子或载体。
在本发明应用较佳的实施方式中,上述重组菌为大肠杆菌或酵母。
本发明还提供了限制性内切酶BamH Ⅰ的突变体的制备方法,将限制性内切酶BamHⅠ的第197位氨基酸从甘氨酸突变为天冬氨酸。
在本发明应用较佳的实施方式中,上述制备方法包括:先将上述的载体转化目的细菌或酵母的感受态细胞,培养,诱导限制性内切酶BamH Ⅰ的突变体的表达。
在本发明应用较佳的实施方式中,上述制备方法还包括:收集表达限制性内切酶BamH Ⅰ的突变体的菌体,破菌后进行BamH Ⅰ的突变体的蛋白纯化;BamH Ⅰ的突变体的蛋白纯化是采用Ni-IDA 6FF琼脂糖纯化树脂的层析柱进行。
本发明具有以下有益效果:
本发明提供了一种限制性内切酶BamH Ⅰ的突变体,与野生型限制性内切酶BamH Ⅰ相比,在第197位的氨基酸发生了突变。由甘氨酸突变为天冬氨酸。突变后的突变体能直接进行重组表达,表达产物经简单的纯化后可得到大量目标蛋白。因突变体蛋白易于大量制备,后续可用于制作蛋白Marker产品中的特定分子量条带。
附图说明
为了更清楚地说明本发明实施例的技术方案,下面将对实施例中所需要使用的附图作简单地介绍,应当理解,以下附图仅示出了本发明的某些实施例,因此不应被看作是对范围的限定,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他相关的附图。
图1为实施例3所对应Ni亲和层析纯化的蛋白的SDS-PAGE效果图,M:proteinMarker;1:上样液;2:流穿液;3:50mmol/L Tris冲洗;4:50mmol/L Tris+50mmol/L咪唑洗脱;5:50mmol/L Tris+100mmol/L咪唑洗脱;6:50mmol/L Tris+200mmol/L咪唑洗脱;7:50mmol/L Tris+500mmol/L咪唑洗脱。
具体实施方式
现将详细地提供本发明实施方式的参考,其一个或多个实例描述于下文。提供每一实例作为解释而非限制本发明。实际上,对本领域技术人员而言,显而易见的是,可以对本发明进行多种修改和变化而不背离本发明的范围或精神。例如,作为一个实施方式的部分而说明或描述的特征可以用于另一实施方式中,来产生更进一步的实施方式。
本发明提供了一种限制性内切酶BamH Ⅰ的突变体,其具有如SEQ ID NO.1所示的氨基酸序列。
发明人在合成限制性内切酶BamH Ⅰ的基因时,发现将编码限制性内切酶BamH Ⅰ的基因的第590位由G突变为A后,会导致翻译后的氨基酸序列第197位由甘氨酸(Glycine,G)突变为天冬氨酸(Aspartic acid,D)。突变获得的突变体经过酶切实验验证,该突变体失去了切割特定DNA序列的功能。进一步将其突变体重组表达后,经过亲和层析纯化。
在其他实施方式中,只要能使得氨基酸序列的第197位由甘氨酸突变为天冬氨酸,包括不限于通过基因突变的方式使得基因的第590位由G突变为A。
本领域技术人员容易想到通过本领域常规的转基因技术、基因编辑技术(如通过锌指核酸内切酶(ZFN,zinc-finger nucleases)技术、类转录激活因子效应物核酸酶(TALEN,transcription activator-like effector nucleases)技术或CRISPR/Cas9)等对目标序列进行改造,使其具有编码如上限制性内切酶BamH Ⅰ的基因,进而获得缺失切割DNA序列功能的限制性内切酶BamH Ⅰ的突变体。
本发明还提供了一种核酸分子,其编码上述限制性内切酶BamH Ⅰ的突变体。
核酸分子可以是质粒或DNA片段。
在本发明应用较佳的实施方式中,上述核酸分子具有SEQ ID NO.2所示的核苷酸序列。
SEQ ID NO.2如下所示:
atggaggttgagaaagagtttattaccgacgaagcgaaggagctgctgtctaaagacaagctgattcagcaggcgtacaacgaggttaaaacgtctatctgctccccgatctggccggcgacctctaagaccttcacgatcaacaacaccgaaaagaactgcaacggcgtcgttccaatcaaggaactgtgctacaccctgctggaagacacctacaactggtaccgcgaaaaaccgctggacatcctgaaactggaaaaaaagaaaggcggtccgatcgacgtgtacaaggagttcatcgagaactctgagctgaaacgtgtgggcatggaatttgaaaccggcaacatctcttctgcgcaccgttctatgaacaaactgctgctgggtctgaaacacggtgagatcgacctggcgatcattctgatgccgatcaaacagctggcgtactacctgacggaccgtgtaaccaatttcgaagaactcgagccgtacttcgaactgaccgaaggtcagccgttcatcttcatcggcttcaacgcggaagcgtataacagcaacgttccgctgatccctaaaggttctgacgacatgtccaagcgcagcatcaaaaagtggaaagataaggtcgaaaacaaa。
本发明还提供了一种载体,其含有上述的核酸分子。
本发明还提供了一种重组菌或重组细胞,其含有上述的核酸分子或载体。
在本发明应用较佳的实施方式中,上述重组菌为大肠杆菌或酵母。
在一种可选的实施方式中,重组细胞为感受态细胞。
本发明还提供了限制性内切酶BamH Ⅰ的突变体、核酸分子、载体或重组菌或重组细胞在制备蛋白Marker中的蛋白标准物中的应用。
本发明还提供了限制性内切酶BamH Ⅰ的突变体的制备方法,将限制性内切酶BamHⅠ的第197位氨基酸从甘氨酸突变为天冬氨酸。
在本发明应用较佳的实施方式中,上述制备方法包括:先将上述的载体转化目的细菌或酵母的感受态细胞,培养,诱导限制性内切酶BamH Ⅰ的突变体的表达。
在本发明应用较佳的实施方式中,上述制备方法还包括:收集表达限制性内切酶BamH Ⅰ的突变体的菌体,破菌后进行BamH Ⅰ的突变体的蛋白纯化;BamH Ⅰ的突变体的蛋白纯化是采用Ni-IDA 6FF琼脂糖纯化树脂的层析柱进行。
为使本发明实施例的目的、技术方案和优点更加清楚,下面将对本发明实施例中的技术方案进行清楚、完整地描述。实施例中未注明具体条件者,按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者,均为可以通过市售购买获得的常规产品。
除非另外指明,否则实践本发明将采用植物生理学、植物分子遗传学、细胞生物学、分子生物学(包含重组技术)、微生物学、生物化学和免疫学的常规技术,所述常规技术在本领域技术人员的能力范围内。文献中充分解释了这种技术,如《分子克隆:实验室手册(Molecular Cloning:A Laboratory Manual)》,第二版(Sambrook等人,1989);分子克隆实验指南(第三版)》(J.萨姆布鲁克等著,2003);《寡核苷酸合成(OligonucleotideSynthesis)》(M.J.Gait编,1984);《植物生理学》(苍晶等人,2017);《酶学方法(Methods inEnzymology)》(学术出版社有限公司(Academic Press,Inc.);《实验免疫学手册(Handbookof Experimental Immunology)》(D.M.Weir和C.C.Blackwell编);《当代分子生物学方法(Current Protocols in Molecular Biology)》(F.M.Ausubel等人编,1987);《植物分子遗传学》(Monica A.Hughes等人著);《PCR:聚合酶链反应(PCR:The Polymerase ChainReaction)》(Mullis等人编,1994),所述文献中的每个文献均通过引用明确并入本文中。
以下结合实施例对本发明的特征和性能作进一步的详细描述。
实施例1
本实施例提供了BamH Ⅰ突变体蛋白表达载体的构建方法。
分别以BamH Ⅰ-F(5’-ggaattcCATATGgaggttgagaaagag-3’,下划线为Nde I酶切位点)为上游引物,以BamH Ⅰ-R(5’-cccAAGCTTatcatttgttttcgaccttatctttccac-3’,下划线为HindⅢ酶切位点)为下游引物,质粒pUC57-BamH Ⅰ G197D为模板,PCR扩增BamH Ⅰ G197D基因。PCR产物经回收后用Nde I和Hind Ⅲ双酶切,双酶切产物回收后连入经过同样双酶切的商品化质粒pET-28a中,得到28a-BamH Ⅰ G197D载体,送测序验证正确。
测序序列如下:
tgggttgtggcggtttcccccttccttgaataattttgtttactttaagaagggagatataccatgggcagcagccatcatcatcatcatcacagcagcggcctggtgccgcgcggcagccatatggaggttgagaaagagttt attaccgacgaagcgaaggagctgctgtctaaagacaagctgattcagcaggcgtacaacgaggttaaaacgtcta tctgctccccgatctggccggcgacctctaagaccttcacgatcaacaacaccgaaaagaactgcaacggcgtcgt tccaatcaaggaactgtgctacaccctgctggaagacacctacaactggtaccgcgaaaaaccgctggacatcctg aaactggaaaaaaagaaaggcggtccgatcgacgtgtacaaggagttcatcgagaactctgagctgaaacgtgtgg gcatggaatttgaaaccggcaacatctcttctgcgcaccgttctatgaacaaactgctgctgggtctgaaacacgg tgagatcgacctggcgatcattctgatgccgatcaaacagctggcgtactacctgacggaccgtgtaaccaatttc gaagaactcgagccgtacttcgaactgaccgaaggtcagccgttcatcttcatcggcttcaacgcggaagcgtata acagcaacgttccgctgatccctaaaggttctgacgacatgtccaagcgcagcatcaaaaagtggaaagataaggt cgaaaacaaatgataagcttgcggccgcactcgagcaccaccaccaccaccactgagatccggctgctaatcaaagcccggaaaaggaagcttattt。
下标线表示BamH Ⅰ G197D基因序列。
实施例2
本实施例提供了BamH Ⅰ突变体蛋白表达的方法。
将实施例1中的表达载体28a-BamH Ⅰ G197D转化大肠杆菌BL21(DE3)感受态细胞,涂布LB平板(含卡那霉素50mg/L),37℃恒温箱过夜培养。
从过夜培养的LB平板上挑取单个菌落到装有50mL LB培养基的250mL摇瓶中,置于37℃恒温摇床,200rpm,培养20h作为种子液。
将培养好的种子液接种到装有100mL自诱导培养基的1000mL摇瓶中,共接种8瓶,每瓶的接种量为5%(v/v),置于恒温摇床,200rpm,37℃培养4h后转为16℃培养20h。其中,自诱导培养基的配方为:乳糖2g/L;蛋白胨10g/L;酵母粉5g/L;NaCl 10g/L;甘油8ml/L。
收集所有培养液,离心后去上清得到菌体。
实施例3
本实施例提供了BamH Ⅰ突变体蛋白的纯化方法。
在实施例2收集菌体的离心瓶中加入200mL含300mmol/L NaCl的PBS缓冲液,使菌体混悬,用高压均质机破碎细胞至混悬液变澄清。将破菌液离心(8000rpm,10min),取上清液备用。
将装有20mL Ni-IDA 6FF琼脂糖纯化树脂的层析柱用含300mmol/L NaCl的PBS缓冲液平衡,之后将上述离心后的上清液上样,然后用Tris(50mmol/L,pH8.0)缓冲液冲洗层析柱,再用含有不同浓度咪唑的Tris(50mmol/L,pH8.0)缓冲液梯度洗脱,其中咪唑浓度依次为50mmol/L;100mmol/L;200mmol/L;500mmol/L。收集洗下来的各组分,各加入loadingbuffer制成上样样品,SDS-PAGE凝胶电泳。
SDS-PAGE效果图参照图1所示。图1显示,本实施例成功纯化出限制性内切酶BamHⅠ的突变体。
以上所述仅为本发明的优选实施例而已,并不用于限制本发明,对于本领域的技术人员来说,本发明可以有各种更改和变化。凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
序列表
<110> 生工生物工程(上海)股份有限公司
<120> 一种限制性内切酶BamHⅠ的突变体及其应用
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atggaggttg agaaagagtt tattaccgac gaagcgaagg agctgctgtc taaagacaag 60
ctgattcagc aggcgtacaa cgaggttaaa acgtctatct gctccccgat ctggccggcg 120
acctctaaga ccttcacgat caacaacacc gaaaagaact gcaacggcgt cgttccaatc 180
aaggaactgt gctacaccct gctggaagac acctacaact ggtaccgcga aaaaccgctg 240
gacatcctga aactggaaaa aaagaaaggc ggtccgatcg acgtgtacaa ggagttcatc 300
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cgcagcatca aaaagtggaa agataaggtc gaaaacaaa 639
Claims (9)
1.一种限制性内切酶BamH Ⅰ的突变体,其特征在于,其具有如SEQ ID NO.1所示的氨基酸序列。
2.一种核酸分子,其特征在于,其编码权利要求1所述的限制性内切酶BamH Ⅰ的突变体。
3.根据权利要求2所述的核酸分子,其特征在于,其具有SEQ ID NO.2所示的核苷酸序列。
4.一种载体,其特征在于,其含有权利要求2-3任一项所述的核酸分子。
5.一种重组菌或重组细胞,其特征在于,其含有权利要求2-3任一项所述的核酸分子或权利要求4所述的载体。
6.根据权利要求5所述的重组菌或重组细胞,其特征在于,所述重组菌为大肠杆菌或酵母。
7.如权利要求1所述的限制性内切酶BamH Ⅰ的突变体的制备方法,其特征在于,将限制性内切酶BamH Ⅰ的第197位氨基酸从甘氨酸突变为天冬氨酸。
8.根据权利要求7所述的制备方法,其特征在于,所述制备方法包括:先将权利要求5所述的载体转化目的细菌或酵母的感受态细胞,培养,诱导限制性内切酶BamH Ⅰ的突变体的表达。
9.根据权利要求8所述的制备方法,其特征在于,所述制备方法还包括:收集表达限制性内切酶BamH Ⅰ的突变体的菌体,破菌后进行BamH Ⅰ的突变体的蛋白纯化;所述BamH Ⅰ的突变体的蛋白纯化是采用Ni-IDA 6FF琼脂糖纯化树脂的层析柱进行。
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