CN114057861A - 一种靶向UBE2C的bio-PROTAC人工蛋白 - Google Patents
一种靶向UBE2C的bio-PROTAC人工蛋白 Download PDFInfo
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- CN114057861A CN114057861A CN202111386453.9A CN202111386453A CN114057861A CN 114057861 A CN114057861 A CN 114057861A CN 202111386453 A CN202111386453 A CN 202111386453A CN 114057861 A CN114057861 A CN 114057861A
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Abstract
本发明属于生物技术领域,涉及一种靶向UBE2C的新型人工蛋白及它的实际应用。本发明的融合蛋白由两个蛋白质结构域WHB结构域和NEL结构域构成,其中WHB结构域来源于与UBE2C有直接相互作用的天然APC2蛋白质的结构域;NEL来自于志贺氏菌的E3酶IPAN9.8的保守E3酶结构域。本发明的bio‑PROTAC能够特异性的识别UBE2C,在无细胞环境中成功实现UBE2C蛋白的泛素化修饰,并能在细胞内降解外源表达的UBE2C。
Description
技术领域
本发明属于生物技术领域,涉及一种靶向UBE2C的新型人工蛋白、其制备方法以及实际应用。
背景技术
UBE2C(Gene ID:11065)是后期促进复合物/环体(APC/C)的专属E2酶(泛素结合酶),主要负责APC/C底物蛋白上Lys-11(K11)上泛素链的起始,之后由APC/C和另一种E2酶UBE2S进一步延长泛素链,从而共同调节有丝分裂进程。目前的研究表明,UBE2C的过度表达会导致染色体错误分离,进而改变细胞周期过程,促进细胞增殖。在许多人类肿瘤组织中,UBE2C被检测出有过度表达,并且这一现象与多种肿瘤的进展和预后不良有关,这些证据说明它参与了肿瘤的发展和侵袭,是一个潜在的癌症靶点,但由于这一蛋白表面比较光滑,导致开发它的抑制剂比较困难,所以目前还没有针对它的小分子抑制剂。
寻找能高效的,特异性的抑制剂是化学生物学领域中长期令人非常感兴趣的方向之一,因为蛋白质是基因的表达产物,也是绝大部分生命活动的直接执行者。针对抑制蛋白,降解蛋白质的方法也是一种思路,其中PROTAC(靶向诱导蛋白降解联合体)是目前最有前景的技术。相比于以往蛋白质相互作用的抑制剂而言,它具有所需剂量更小,活性更高,能靶向一些以前不可成药的靶点等优势。
现在的主流化学小分子PROTAC一般由三部分构成,其中一端是能特异性靶向目标蛋白的配体(通常是小分子抑制剂),另一端是E3泛素连接酶的共价连接配体,中间会有一段linker将二者连接起来。一旦与目的蛋白结合,PROTAC就可以招募E3对其进行泛素化,最终通过“泛素-蛋白酶体”途径被降解。
发明内容
本发明要解决的技术问题是提供后期促进复合物/环体(APC/C)的专属E2酶(泛素结合酶)的抑制剂,通过靶向诱导蛋白降解联合体的方式招募E3对其进行泛素化,最终通过“泛素-蛋白酶体”途径降解UBE2C。
成功构建PROTAC分子的一个关键点在于如何高效的筛选出能与靶蛋白结合的配体,这常常是一个费时费力的过程。与传统的PROTAC不同,bio-PROTAC本质上是一个融合蛋白,其中一端是能与目标蛋白结合的蛋白质结构域,另一端则是一个E3酶结构域。就目前而言,大部分的bio-PROTAC是基于目的蛋白的纳米抗体构建的,一些成功的例子包括靶向GFP,PCNA和K-RAS的bio-PROTAC。但由于UBE2C是一个潜在的,相关研究还不多的癌症靶点,因此没有它的纳米抗体被报道,所以无法按照构建bioPROTAC的一般思路去进行。同时,由于这一蛋白表面比较的光滑,因此开发相应的化学抑制剂也比较困难,所以进行传统的PROTAC的开发也缺乏切入点。
本发明中,基于bio-PROTAC这一体系开发了一个靶向UBE2C的人工融合蛋白。该人工融合蛋白借助WHB结构域靶向UBE2C(WHB是属于后期促进复合物APC/C的APC2亚基的一个结构域),之后NEL结构域会在靶蛋白氨基酸残基上连上泛素并进一步延伸成泛素链。该人工蛋白可以在无细胞体系中实现UBE2C的泛素化修饰,并能在细胞中达到降解外源表达的UBE2C的效果。
本发明在上述研究基础上完成。
一方面,本发明提供了一种靶向UBE2C的bio-PROTAC人工蛋白(即UBE2C体外泛素化雷达)。所述的蛋白UBE2C体外泛素化雷达由两个蛋白质结构域WHB结构域和NEL结构域构成,其中WHB结构域来源于与UBE2C有直接相互作用的天然APC2蛋白质的结构域;NEL来自于志贺氏菌的E3酶IPAH9.8的保守E3酶结构域,IpaH家族蛋白是一类来源于革兰氏阴性菌的E3泛素连接酶,IPAH9.8的完整结构包含三个部分,分别是N端的T3SS信号序列、负责结合底物的LRR结构域、以及具有E3酶功能的C端保守的NEL结构域。这类E3酶能够通过劫持宿主的泛素-蛋白酶体信号通路来抑制宿主炎症和内源性免疫应答反应,从而加速感染的过程。相比于大部分哺乳动物细胞内源性的E3酶来说,它的结构更加简单,没有复杂的翻译后修饰,更容易使用原核细菌表达从而进行体外活性的探索。
可选的,WHB结构域位于N端,NEL结构域位于C端,二者之间含有连接物(linker),连接物由10-15氨基酸残基组成,其序列选自:
以S、G、N为主的氨基酸序列;或者-GQQNTLHRPLA-(SEQ ID NO 1),-SSGSSGSSG-(SEQ ID NO 2),-SSGSSGSSGSSG-(SEQ ID NO 3),-SSGSSGSSGSSGSSG-(SEQ ID NO4),-NSSSNNNNNNN-(SEQ ID NO 5),-NSSSNNNNNNNNNNLG-(SEQ ID NO 6),-SSGNNNNNNSSG-(SEQID NO 7),-NNNSSGNNNSSG-(SEQ ID NO 8),-SSGGQQNTLHRPLASSG-(SEQ ID NO9),-GQQNTLHRPLANNNSSG-(SEQ ID NO 10)中的任意一种。
可选的,WHB结构域来自APC2蛋白(Gene ID:29882)的S732-S822;或者NEL结构来自IPAH9.8(Gene ID:1238048)的G245-S545。本发明也包括根据本领域的常规技术在此基础上的常规变通。
可选的,本发明的靶向UBE2C的bio-PROTAC,其结构通式如图6所示。其中,targetprotein binding domain为来自APC2蛋白的WHB结构域(S732-S822),约10.4kDa,E3是来自IPAH9.8的E3泛素连接酶结构域(G245-S545),约34.4kDa,中间的黑线为linker。
在本发明的一个优选例中,所述的靶向UBE2C的bio-PROTAC人工蛋白的序列如下所示:
SDDESDSGMASQADQKEEELLLFWTYIQAMLTNLESLSLDRIYNMLRMFVVTGPALAEIDLQELQGYLQKKVRDQQLVYSAGVYRLPKNCS-GQQNTLHRPLA-DAVTAWFPENKQSDVSQIWHAFEHEEHANTFSAFLDRLSDTVSARNTSGFREQVAAWLEKLSASAELRQQSFAVAADATESCEDRVALTWNNLRKTLLVHQASEGLFDNDTGALLSLGREMFRLEILEDIARDKVRTLHFVDEIEVYLAFQTMLAEKLQLSTAVKEMRFYGVSGVTANDLRTAEAMVRSREENEFTDWFSLWGPWHAVLKRTEADRWAQAEEQKYEMLENEYPQRVADRLKASGLSGDADAEREAGAQVMRETEQQIYRQLTDEVLALRLSENGSQLHHS(SEQ ID NO 11)。
本发明还包括表达上述融合蛋白的所需基因序列。本发明提供了一种核酸,该核酸是上述靶向UBE2C的bio-PROTAC人工蛋白的编码序列。其核酸序列为:5’-agtgacgacgagagcgactccggcatggcctcccaggccgaccagaaggaggaggagctgctgctcttctggacgtacatccaggccatgctgaccaacctggagagcctctcactggatcgtatctacaacatgctccgcatgtttgtggtgactgggcctgcactggccgagattgacctgcaggagctgcagggctacctgcagaagaaggtgcgggaccagcagctcgtctactcggccggcgtctaccgcctgcccaagaactgcagcGGCCAGCAGAACACACTCCACAGACCACTCGCCGACGCCGTGACAGCCTGGTTCCCTGAGAACAAGCAGTCTGACGTGTCCCAGATTTGGCACGCCTTCGAGCACGAGGAGCACGCCAACACATTCTCTGCCTTCCTCGACCGGCTCTCTGACACAGTGTCTGCCCGCAACACATCCGGCTTCAGGGAGCAGGTGGCCGCCTGGCTGGAGAAGCTGTCTGCCTCTGCCGAATTAAGGCAGCAGTCTTTCGCCGTGGCCGCCGACGCCACAGAGTCTTGCGAGGACCGCGTGGCCCTCACATGGAACAACCTCCGCAAGACACTGCTCGTGCACCAGGCCTCTGAGGGCCTGTTCGACAACGACACCGGCGCCCTCCTGTCCCTGGGCAGGGAGATGTTCAGACTGGAGATCCTGGAGGACATTGCACGGGACAAGGTGCGCACCCTCCACTTCGTGGACGAGATTGAGGTGTACCTCGCCTTCCAGACCATGCTCGCCGAGAAGTTACAGCTGTCTACAGCCGTGAAGGAGATGCGCTTCTACGGCGTGTCCGGCGTGACAGCCAACGACCTGCGGACAGCCGAGGCAATGGTGCGGAGCAGAGAGGAGAACGAGTTCACAGACTGGTTCTCCCTGTGGGGCCCTTGGCACGCCGTGCTGAAGCGGACCGAGGCCGACCGCTGGGCCCAGGCCGAGGAGCAGAAGTACGAGATGCTGGAGAACGAGTACCCCCAGCGGGTGGCCGACAGACTCAAGGCCAGCGGCCTGTCCGGCGACGCCGACGCCGAGCGGGAGGCCGGCGCCCAGGTGATGCGCGAGACAGAGCAGCAGATTTACCGGCAGCTCACCGACGAGGTGCTCGCCCTCAGACTGTCTGAGAACGGCTCTCAGCTCCACCACTCT-3’(SEQ ID NO 12)。
另一方面,本发明包括上述靶向UBE2C的bio-PROTAC人工蛋白UBE2C体外泛素化雷达的制备方法。本发明的融合蛋白可以采用蛋白合成的常规方法,例如根据目标蛋白的序列逐个连接合成,也可以分段表达或者合成蛋白,再连接。例如,可以如冷泉港的《分子克隆》所示例,包括目标蛋白表达载体构建和Bio-PROTAC的诱导表达步骤。
本发明提供了利用原核系统表达纯化该蛋白的技术路线。可选用的原核表达系统可以包括是常规的原核表达载体。
可选的,原核表达载体构建包括:将bio-PROTAC人工蛋白的编码序列为模板进行扩增得到带有同源序列的插入片段,将线性化载体和插入片段按比例混合,转入感受态细胞中,获得目的基因正确插入的原核表达载体。
Bio-PROTAC的诱导表达包括:将构建好的原核表达载体利用热激法转入同样的感受态细胞中,后从平板上挑取一个单克隆于含卡那霉素抗性LB的培养基中培养过夜;次日将菌液转移至含LB培养基的锥形瓶中培养,待OD值到0.4-0.6时加入诱导剂IPTG使其终浓度为0.25mM,之后在16℃培养过夜。
可选的,所述的制备方法还包括bio-PROTAC纯化步骤。
在本发明的一个优选实施例中,所述bio-PROTAC人工蛋白的制备方法具体包括以下步骤:
步骤(1)的原核表达载体构建包括但不限于使用pet28系列原核载体表达:将pet28a反向PCR以获得线性载体,将线性化载体末端15bp-20bp序列作为同源序列,并将其分别添加到基因特异性正/反向扩增引物序列的5’端,之后以目的基因为模板进行扩增得到带有同源序列的插入片段;将线性化载体和插入片段按比例混合,将线性模板与插入片段按摩尔比1:2混合后在介入1μl Exnase II,2μl 5×CE II Buffer,用dd H2O补齐至10μl,于37℃反应30min从而完成重组反应,之后将10μl反应液全部加入DH5a感受态中,在冰上孵育30min后,于42℃热激90s,再继续在冰上孵育2min,加入200μl无抗性的LB培养基于37℃恒温摇床培养1h后涂板,第二天挑取单克隆提取质粒并测序验证,最终获得目的基因正确插入的原核表达载体;
步骤(2)的Bio-PROTAC的诱导表达包括但不限于利用E.COLI BL21系列菌株进行表达:将构建好的原核表达载体利用和上一步一样的热激法转入E.COLI BL21感受态,之后从平板上挑取一个单克隆于5ml含卡那霉素抗性LB的培养基中37℃培养过夜;次日将5ml菌液全部转移至含1L LB培养基的锥形瓶中培养,待OD值到0.4-0.6时加入诱导剂IPTG使其终浓度为0.25mM,之后在16℃培养过夜;或者
步骤(3)的Bio-PROTAC纯化包括但不限于:
将诱导过夜的菌液5000rpm离心10min,弃掉上清,菌体用100ml纯化缓冲液重悬,运用均质机使菌体破碎,破碎后的菌液在18000rpm离心60min取上清,之后借助载体上自带的6*组氨酸标签,可以使用镍离子-组氨酸亲和层析柱进行初步纯化,接着,再用HiLoad16/600Superdex 200pg层析柱将前一步亲和层析纯化所得的蛋白进行了二次纯化,所得的纯蛋白质超滤浓缩至20mg/ml,分装并保存在50mM Tris 8.0,500mM Nacl,2mMβ-ME的缓冲液中。
再一方面,本发明还包括所述靶向UBE2C的bio-PROTAC人工蛋白UBE2C体外泛素化雷达的应用。
本发明的一个实施例借助SDS变性凝胶电泳实现了所述靶向UBE2C的bio-PROTAC人工蛋白可以在无细胞条件下对UBE2C的泛素化修饰的可行性验证。本发明的另一个实施例提供了该人工蛋白降解外源转入细胞的UBE2C的可行性验证。
使用所述的bio-PROTAC人工蛋白,能够特异性识别或者降解UBE2C。需注意的是,本发明的靶向UBE2C的bio-PROTAC人工蛋白无法降解细胞内源表达的UBE2C。我们认为之所以造成这样的结果是因为WHB与UBE2C之间的亲和力较弱所导致的,假设能将用于靶向UBE2C的部分替换成相应的纳米抗体,我们认为这一情况可以得到解决。但遗憾的是,正如前文所述,目前还没有针对这一蛋白的纳米抗体。
可选的,所述靶向UBE2C的bio-PROTAC人工蛋白可以在体外无细胞环境中实现UBE2C蛋白的泛素化修饰。
本发明公开了一种靶向UBE2C的bio-PROTAC的具体设计和其在使UBE2C蛋白泛素化中的应用。本发明中的bio-PROTAC由两个蛋白质结构域构成,其中WHB结构域来源于并证明与UBE2C有直接相互作用的天然APC2蛋白质的结构域;NEL则是来自于志贺氏菌的E3酶IPAN9.8的保守E3酶结构域。本发明的bio-PROTAC能够特异性的识别UBE2C,在无细胞环境中成功实现UBE2C蛋白的泛素化修饰,并能在细胞内降解外源表达的UBE2C。
附图说明
为了更清楚地说明本申请实施例中的技术方案,下面将对实施例中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图仅仅是本申请的一些实施例,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的附图。
图1是bio-PROTAC使目标蛋白泛素化原理示意图及bio-PROTAC融合蛋白结构示意图。
在体外实验中,该融合蛋白UBE2C体外泛素化雷达借助WHB结构域靶向UBE2C,之后NEL结构域会行使其E3泛素连接酶的功能,在人为加入的E1酶,E2酶,以及泛素和ATP的帮助下,于靶蛋白UBE2C的赖氨酸残基上连接上泛素并进一步延伸成泛素链,进而完成对UBE2C的泛素化修饰;而在细胞内,理论上来说若WHB-NEL能顺利与UBE2C结合,则NEL可以利用宿主自身的泛素化系统来进行泛素化修饰。
在bio-PROTAC融合蛋白结构示意图中,所述的蛋白由两个蛋白质结构域WHB结构域和NEL结构域构成。
图2是bio-PROTAC氨基酸序列。
其中,下划线标注部分为linker。linker可含有以下所列的氨基酸序列或其他常见的以S,G,N为主的由10-15氨基酸残基:-GQQNTLHRPLA-(SEQ ID NO 1),-SSGSSGSSG-(SEQID NO 2),-SSGSSGSSGSSG-(SEQ ID NO 3),-SSGSSGSSGSSGSSG-(SEQ ID NO 4),-NSSSNNNNNNN-(SEQ ID NO 5),-NSSSNNNNNNNNNNLG-(SEQ ID NO 6),-SSGNNNNNNSSG-(SEQID NO 7),-NNNSSGNNNSSG-(SEQ ID NO 8),-SSGGQQNTLHRPLASSG-(SEQ ID NO 9),-GQQNTLHRPLANNNSSG-(SEQ ID NO 10)。
图3是bio-PROTAC的原核系统表达及纯化。
其中,上图右侧的蛋白电泳图中,从左到右分别是supernatant上清液、precipitation沉淀、flow throw流穿液、elution洗脱液和蛋白分子量标记marker。
图4是野生型bio-PROTAC和突变体(Cys to Ala)在体外无细胞环境中对UBE2C进行泛素化修饰的活性验证。
其中,第7泳道样品即60分钟的最后一个样品,箭头所指条带表明底物蛋白分别为被修饰上一条或两条泛素链的底物蛋白,相比之下,在1-8泳道内没有任何一个其他泳道出现这样的泛素化条带。此外,第9和10泳道为突变体bio-PROTAC(本发明所提供的bio-PROTAC蛋白序列第184位的半胱氨酸突变为丙氨酸,这导致了NEL结构域无法行使E3泛素连接酶的功能),可以看见在用与第7道完全相同的条件和试剂处理后泛素化修饰的蛋白条带没有出现,说明突变体已无法使UBE2C泛素化,证明本发明中原始bio-PROTAC蛋白序列在使UBE2C泛素化中的必要性。
图5是bio-PROTAC可以在细胞中降解外源表达的UBE2C。
图6是靶向UBE2C的bio-PROTAC的结构示意图。
其中,target protein binding domain为来自APC2蛋白的WHB结构域(S732-S822),约10.4kDa,E3是来自IPAH9.8的E3泛素连接酶结构域(G245-S545),约34.4kDa,中间的黑线为linker。
具体实施方式
本发明提供了一种靶向UBE2C的bio-PROTAC,它的N端是来自APC2蛋白的WHB结构域(S732-S822),C端是来自IPAH9.8的NEL结构域(G245-S545),二者中间可含有以下所列的氨基酸序列或其他常见的以S,G,N为主的由10-15氨基酸残基组成的序列做为linker。
下面将对本申请实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本申请一部分实施例,而不是全部的实施例。基于本申请中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本申请保护的范围。
实施例1.bio-PROTAC的原核表达及分离纯化
本发明bio-PROTAC的原核表达及分离纯化步骤如下:
(1)原核表达载体构建:
该bio-PROTAC可以使用pet28系列原核载体表达。首先,将pet28a反向PCR以获得线性载体。将线性化载体末端15bp-20bp序列作为同源序列,并将其分别添加到基因特异性正/反向扩增引物序列的5’端,之后以目的基因为模板进行扩增得到带有同源序列的插入片段。将线性化载体和插入片段按比例混合,将线性模板与插入片段按摩尔比1:2混合后在介入1μlExnase II,2μl 5×CE II Buffer,用dd H2O补齐至10μl,于37℃反应30min从而完成重组反应。之后将10μl反应液全部加入DH5a感受态中,在冰上孵育30min后,于42℃热激90s,再继续在冰上孵育2min,加入200μl无抗性的LB培养基于37℃恒温摇床培养1h后涂板,第二天挑取单克隆提取质粒并测序验证,最终获得目的基因正确插入的原核表达载体。
(2)Bio-PROTAC的诱导表达:
本bio-PROTAC适合利用E.COLI BL21系列菌株进行表达。将构建好的原核表达载体利用和上一步一样的热激法转入E.COLI BL21感受态。之后从平板上挑取一个单克隆于5ml含卡那霉素抗性LB的培养基中37℃培养过夜。次日将5ml菌液全部转移至含1L LB培养基的锥形瓶中培养,待OD值到0.4-0.6时加入诱导剂IPTG使其终浓度为0.25mM,之后在16℃培养过夜。
(3)Bio-PROTAC纯化:
将诱导过夜的菌液5000rpm离心10min,弃掉上清,菌体用100ml纯化缓冲液(50mMTris 8.0,500mM Nacl,20mM Imidazole)重悬,运用均质机使菌体破碎,破碎后的菌液在18000rpm离心60min取上清。之后借助载体上自带的6*组氨酸标签,可以使用镍离子-组氨酸亲和层析柱进行初步纯化,接着,再用HiLoad 16/600Superdex 200pg层析柱将前一步亲和层析纯化所得的蛋白进行了二次纯化,所得的纯蛋白质超滤浓缩至20mg/ml,分装并保存在50mM Tris 8.0,500mM Nacl,2mMβ-ME的缓冲液中。
实施例2.体外泛素化活性测定
将0.25μM E1(鼠源UBA1,120Kd),2μM E2(UBE2D2,19.6Kd),0.5μM E3(bio-PROTACWT,bio-PROTACCA,43.5Kd),50μM Ub(人源Ub,8.6Kd),5mM MgCl,2.5mM ATP和2MmUBE2C(20Kd)混合,在室温下于PBS缓冲液(pH7.4)中孵育。在不同时间点取样,把所取的反应混合物添加到同等体积的2*SDS-PAGEloading中,在沸水浴中加热10min。之后用12%SDS-聚丙烯酰胺凝胶进行电泳,电泳过后的凝胶用考马斯亮蓝染色液染色,之后在含醋酸脱色液中过夜脱色。最后用凝胶成像仪对凝胶进行成像。
结果如图4所示,正确序列的bio-PROTAC(野生型)能够在体外使UBE2C泛素化,而突变体(本专利所提供的bio-PROTAC蛋白序列第184位的半胱氨酸突变为丙氨酸)由于关键活性氨基酸被突变因而无法进一步执行E3泛素连接酶功能,则不能使UBE2C泛素化。
需要说明的是,本发明是第一个靶向UBE2C的bio-PROTAC,也是第一个以WHB-linker-NEL为主体结构进行设计的PROTAC,虽然它未能如最初所设想的那样成功降解内源的UBE2C,但克服了长久以来困扰本领域研究者筛选不到合适的能与靶蛋白结合的配体的难题,为后续靶向UBE2C的bioPROTAC的开发,以及针对其他潜在癌症靶标的bioPROTAC的开发时如何选择靶蛋白的配体或者是选择何种E3酶提供了一个良好的基础。
实施例3.bio-PROTAC在细胞内降解外源性UBE2C的活性验证
(1)真核表达载体构建:由于蛋白本身难以穿过细胞膜,所以需要先构建真核细胞表达载体。构建方式与上文所述原核表达载体构建方式类似,此处不再赘述。本bio-PROTAC可以利用PCDNA3.1系列载体构建。
(2)真核载体转染进入细胞:转染前一日用不含抗生素培养液接种于6孔板中,第二日待细胞密度生长至70%-90%时进行转染。转染试剂推荐使用lipo 2000(thermofisher)。转染前应先将细胞培养液更换为opti-MEM。转染时,首先将DNA和8μllipo 2000分别用250μl opti-MEM稀释,5min后将二者混合再共孵育20min,之后把脂质体-DNA混合液缓慢加入细胞培养液中。由于是针对外源UBE2C的降解,因此需要同时转入表达UBE2C的表达载体。
(3)降解活性验证:转染24h可收样进行western blot验证外源UBE2C是否降解。具体过程为使用高效RIPA裂解液和蛋白酶抑制剂组成的裂解缓冲液裂解细胞液。裂解液用13000rpm离心10min后取上清,运用Braford试剂盒(碧云天)进行定量,每孔裂解液取出20ng蛋白样品,加入等体积2*SDS-PAGE loading制样。样品用12%SDS-PAGE凝胶进行分析,电泳完成后于220V 1h转移到聚偏二氟乙烯(PVDF)膜上,并用各自的抗体进行免疫印迹。最后用化学发光检测试剂盒进行检测,并用Bio-Rad成像仪成像。
结果显示,如图5所示,bio-PROTAC在细胞内能够成功的降解外源性UBE2C(本例中所用linker为-GQQNTLHRPLA-)。
以上所述,仅为本申请的具体实施方式,但本申请的保护范围并不局限于此,任何熟悉本领域技术的技术人员在本申请公开的技术范围内,可轻易想到的变化或替换,都应涵盖在本申请的保护范围之内。因此,本申请的保护范围应以所述权利要求的保护范围为准。
序列表
<110> 深圳湾实验室坪山生物医药研发转化中心
北京大学
<120> 一种靶向UBE2C的bio-PROTAC人工蛋白
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Claims (10)
1.一种靶向UBE2C的bio-PROTAC人工蛋白,其特征在于,所述的蛋白由两个蛋白质结构域WHB结构域和NEL结构域构成,其中WHB结构域来源于与UBE2C有直接相互作用的天然APC2蛋白质的结构域;NEL来自于志贺氏菌的E3酶IPAN9.8的保守E3酶结构域。
2.根据权利要求1所述的靶向UBE2C的bio-PROTAC人工蛋白,其特征在于,WHB结构域位于N端,NEL结构域位于C端,二者之间含有连接物,连接物由10-15氨基酸残基组成,其序列选自:
以S、G、N为主的氨基酸序列;或者
-GQQNTLHRPLA-,-SSGSSGSSG-,-SSGSSGSSGSSG-,-SSGSSGSSGSSGSSG-,-NSSSNNNNNNN-,-NSSSNNNNNNNNNNLG-,-SSGNNNNNNSSG-,-NNNSSGNNNSSG-,-SSGGQQNTLHRPLASSG-,-GQQNTLHRPLANNNSSG-中的任意一种。
3.根据权利要求1所述的靶向UBE2C的bio-PROTAC人工蛋白,其特征在于,WHB结构域来自APC2蛋白的S732-S822;或者
NEL结构来自IPAH9.8的G245-S545。
4.根据权利要求1所述的靶向UBE2C的bio-PROTAC人工蛋白,其特征在于,所述的靶向UBE2C的bio-PROTAC人工蛋白的序列如下所示:
SDDESDSGMASQADQKEEELLLFWTYIQAMLTNLESLSLDRIYNMLRMFVVTGPALAEIDLQELQGYLQKKVRDQQLVYSAGVYRLPKNCS-GQQNTLHRPLA-DAVTAWFPENKQSDVSQIWHAFEHEEHANTFSAFLDRLSDTVSARNTSGFREQVAAWLEKLSASAELRQQSFAVAADATESCEDRVALTWNNLRKTLLVHQASEGLFDNDTGALLSLGREMFRLEILEDIARDKVRTLHFVDEIEVYLAFQTMLAEKLQLSTAVKEMRFYGVSGVTANDLRTAEAMVRSREENEFTDWFSLWGPWHAVLKRTEADRWAQAEEQKYEMLENEYPQRVADRLKASGLSGDADAEREAGAQVMRETEQQIYRQLTDEVLALRLSENGSQLHHS。
5.一种核酸,其特征在于,该核酸是权利要求1-4中任意一项所述靶向UBE2C的bio-PROTAC人工蛋白的编码序列。
6.权利要求1-4中任意一项所述靶向UBE2C的bio-PROTAC人工蛋白的制备方法,其特征在于,所述的制备方法包括以下步骤:
(1)原核表达载体构建:
将权利要求5所述的核酸为模板进行扩增得到带有同源序列的插入片段,将线性化载体和插入片段按比例混合,转入感受态细胞中,获得目的基因正确插入的原核表达载体;
(2)Bio-PROTAC的诱导表达:
将构建好的原核表达载体利用热激法转入同样的感受态细胞中,后从平板上挑取一个单克隆于含卡那霉素抗性LB的培养基中培养过夜;次日将菌液转移至含LB培养基的锥形瓶中培养,待OD值到0.4-0.6时加入诱导剂IPTG使其终浓度为0.25mM,之后在16℃培养过夜。
7.根据权利要求6所述的制备方法,其特征在于,所述的制备方法还包括bio-PROTAC纯化步骤。
8.根据权利要求6所述的制备方法,其特征在于,
步骤(1)的原核表达载体构建包括但不限于使用pet28系列原核载体表达:将pet28a反向PCR以获得线性载体,将线性化载体末端15bp-20bp序列作为同源序列,并将其分别添加到基因特异性正/反向扩增引物序列的5’端,之后以目的基因为模板进行扩增得到带有同源序列的插入片段;将线性化载体和插入片段按比例混合,将线性模板与插入片段按摩尔比1:2混合后在介入1μlExnase II,2μl 5×CE II Buffer,用dd H2O补齐至10μl,于37℃反应30min从而完成重组反应,之后将10μl反应液全部加入DH5a感受态中,在冰上孵育30min后,于42℃热激90s,再继续在冰上孵育2min,加入200μl无抗性的LB培养基于37℃恒温摇床培养1h后涂板,第二天挑取单克隆提取质粒并测序验证,最终获得目的基因正确插入的原核表达载体;
步骤(2)的Bio-PROTAC的诱导表达包括但不限于利用E.COLI BL21系列菌株进行表达:将构建好的原核表达载体利用和上一步一样的热激法转入E.COLI BL21感受态,之后从平板上挑取一个单克隆于5ml含卡那霉素抗性LB的培养基中37℃培养过夜;次日将5ml菌液全部转移至含1L LB培养基的锥形瓶中培养,待OD值到0.4-0.6时加入诱导剂IPTG使其终浓度为0.25mM,之后在16℃培养过夜;或者
步骤(3)的Bio-PROTAC纯化包括但不限于:
将诱导过夜的菌液5000rpm离心10min,弃掉上清,菌体用100ml纯化缓冲液重悬,运用均质机使菌体破碎,破碎后的菌液在18000rpm离心60min取上清,之后借助载体上自带的6*组氨酸标签,可以使用镍离子-组氨酸亲和层析柱进行初步纯化,接着,再用HiLoad 16/600Superdex 200pg层析柱将前一步亲和层析纯化所得的蛋白进行了二次纯化,所得的纯蛋白质超滤浓缩至20mg/ml,分装并保存在50mM Tris 8.0,500mM Nacl,2mMβ-ME的缓冲液中。
9.权利要求1-4中任意一项所述靶向UBE2C的bio-PROTAC人工蛋白的应用,其特征在于,所述的bio-PROTAC人工蛋白特异性识别或者降解UBE2C。
10.根据权利要求9所述的应用,其特征在于,所述靶向UBE2C的bio-PROTAC人工蛋白在无细胞环境中实现UBE2C蛋白的泛素化修饰。
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