不依赖天然外显子的蛋白质内含子定向进化筛选系统

蛋白质剪接技术为在蛋白质水平上直接对蛋白质进行修饰和加工提供了一种全新的解决方案,因而在蛋白质工程及相关领域具有非常广阔的应用前景。现阶段,大部分天然的蛋白质内含子在异源蛋白质中剪接活性非常低,极大限制了蛋白质内含子的开发和应用。为了开发一个可以同时对蛋白质内含子通用性和剪接活性进行筛选的系统,利用Bsa I限制性内切酶识别位点和切割不重合的特性,将Ter ThyX内含子(不含外显子序列)插入到卡那霉素抗性蛋白基因的多个位点。并且摒弃了以往需要结合天然外显子以实现剪接的方法,可以同时对蛋白质内含子的剪接活性和通用性进行筛选。Western blot结果和卡那霉素平板生长结果表明,通过卡那霉素筛选系统可以精确的将蛋白质内含子剪接反应与卡那霉素抗性结合起来,仅从卡那霉素平板上的菌落生长情况即可完成蛋白质内含子剪接活性阳性突变的筛选,是一个快速,稳定的定向进化筛选系统。     

定向进化提高微小蛋白质内含子Ter DnaE-3 剪接活性的研究

目前,蛋白质内含子在蛋白质工程领域中得到越来越广泛的应用。为提高微小蛋白质内含子Ter DnaE-3(Trichodesmium erythraeum)在异源宿主中的剪接活性,采用易错PCR技术,通过改变反应体系中dNTP、Mg2+、Mn2+的浓度等手段,借助依赖卡那霉素的蛋白质内含子筛选系统进行筛选。Western印迹结果表明:通过定向进化,其中5号突变体的剪接活性从原始的约20%提高至约85%;9号突变体能够避免发生剪接副反应,即N端断裂反应。氨基酸突变位点与剪接活性变化的相关性分析表明:参与α-helix形成的氨基酸的突变极有可能影响蛋白质内含子的断裂反应,参与β-sheet形成的氨基酸的突变则有可能影响蛋白质内含子结构的紧凑性。通过定向进化提高微小蛋白质内含子Ter DnaE-3在异源宿主中的剪接活性,进一步验证依赖卡那霉素抗性的筛选系统的可行性,为扩大蛋白质内含子的应用范围奠定基础。     

DNA展示系统介导的断裂蛋白质内含子体外定向进化方法的研究

蛋白质内含子对于外显子的选择性限制了它的应用范围。目前,已经建立的卡那霉素定向进化系统适用于微小蛋白质内含子,但不适用于断裂蛋白质内含子。为了研究适用于断裂蛋白质内含子的定向进化的方法,我们引入了DNA展示系统。在该系统中,生物素化基因在人工细胞中与其表达的融合蛋白（内含子C端-外显子-生物素结合蛋白）形成DNA 蛋白质连接体。只有能与后续加入的N端蛋白质内含子前体（Flag-内含子N端）发生剪接反应的DNA 蛋白质连接体,才能被加上旗帜标签（Flag）,从而被抗旗帜标签抗体纯化柱（Anti Flag antibody M2 agarose）筛选富集。为了验证该系统的可行性,实验构建了2个基因,即含有具剪接活性的蛋白质内含子的阳性基因IRC和含有不具剪接活性的蛋白质内含子的阴性基因IRCM。实验首先通过Western印迹和琼脂糖凝胶电泳证明,人工细胞中的体外转录翻译系统不仅可高表达500个氨基酸的蛋白质,而且表达的蛋白中内含子仍保持原有的剪接能力。生物素结合蛋白能结合95%的DNA,并且形成的DNA 蛋白质连接体可以被筛选富集,最终证明了该系统用于断裂蛋白质内含子定向进化筛选的可行性。随后,为了检测系统的富集效率,制备了人为的基因“突变库”,即将基因IRC和IRCM以摩尔比1:10的比例混合,经过2轮筛选后,阳性基因IRC可以被10倍富集,进一步证明了体外筛选方法的可行性。该方法为后续针对不同宿主进化出不同的断裂蛋白质内含子提供筛选方法支持,也为断裂蛋白质内含子的在生物技术和研究领域的应用奠定了基础。     

基于卡那霉素抗性建立的研究I类内含子结构与功能关系的系统

Ⅰ类内含子（group I intron）是研究RNAs结构与功能关系的理想元件, 在 解释RNA折叠理论、催化机制等方面起着重要作用;对其结构与功能关系的研究也 因此成为一个非常重要的课题. 本研究建立了一个基于卡那霉素抗性进行Ⅰ类内含子结构与功能关系研究系统,将源于海洋蓝细菌Nostoc punctiforme（Npu）核糖核酸还原酶基因（ribonucleotide reductase,Rir）中的1个Ⅰ类内含子插入到pDrive质粒的卡那霉素抗性基因（kanamycin resistance gene,KanR）内构建得pKR12质粒并转化大肠杆菌（E.coli）. 只有内含子剪接的阳性克隆才能生成 KanR蛋白并在Kan抗性平板上生长. 结果显示,pKR12转入E.coli后不能在Kan抗性 平板上生长, RT-PCR检测仅可见前体带, 表明插入到KanR中的Npu Rir内含子没有发生剪接. 随后通过易错PCR建立内含子的随机突变库并用Kan抗性筛选进行定向演化, 产生有剪接活性的内含子突变体, RT-PCR检测显示剪接发生. 由于内含子剪接活性的改变可通过Kan抗性变化在LB平板上得以反映, 因此该系统有望成为简单快速地研究Ⅰ类内含子结构与功能关系的有利工具.     

PRP8蛋白质反式剪接系统的建立

真菌病原体Cryptococcus neoformansAD血清型剪接体蛋白PRP8蛋白质内含子是目前 发现的第2个存在于真核生物体核基因组中的蛋白质内含子.它的宿主基因prp8编码的PRP 8蛋白作为剪接体的1个组分,是1个高度保守的mRNA剪接蛋白.将组氨酸标签插入克隆自真菌病原体Cryptococcus neoformans AD血清型的PRP8蛋白质内含子中,并将该蛋白质内含子进行人工断裂,获得断裂蛋白质内含子,在大肠杆菌中鉴定其剪接活性.研究结果表明：所获得的改造型蛋白质内含子均表现出高效的剪接活性.利用此Cryptococcus neoformansAD血清型PRP8 断裂蛋白质内含子,成功构建了蛋白质反式剪接系统.这一反式剪接系统可用于其他蛋白质的连接与合成,有望成为蛋白质工程中的一种有用工具.     

蛋白质内含子在特定氨基酸序列中的剪接

基因工程技术已经被广泛应用于抗体的生产。但是由于抗体的分子量较大,导致合成抗体较为困难。蛋白质内含子是前体蛋白质中的一段氨基酸序列,能够将自身剪切出来,并将两端的外显子连接形成成熟的蛋白质。将抗体的Fab(antigen binding fragment)和Fc(crystalline fragment)分别与蛋白质内含子(intein) 的N端(IN)和C端(IC)融合表达,利用蛋白质内含子的剪接功能,可形成完整的抗体分子。KSCDKTH是存在于抗体铰链区(hinge region)的一段氨基酸序列,如果在KSCDKTH序列中筛选到高效剪接的蛋白质内含子,即可通过蛋白质剪接,将抗体分子的Fab和Fc剪接形成完整抗体。本文筛选发现,Ssp DnaX的3种断裂蛋白质内含子(S0, S1, S11)具有在KSCDKTH序列中高效剪接的能力,这一研究结果为抗体的剪接合成提供了可行性。     

高剪接活性断裂蛋白质内含子的体内切割

蛋白质内含子介导的断裂(切割)反应被用于蛋白质纯化、连接和环化等,但目前仍存在断裂效率低、断裂反应的不可控、产物复杂等问题。蛋白质内含子的定点突变可导致其N端或C端断裂。其末位氨基酸突变则剪接反应第3步天冬酰胺环化无法进行,发生N端断裂;其首位氨基酸发生突变则剪接反应第一步酰基重排及其后续步骤均无法进行,而天冬酰胺环化仍可进行,发生C端断裂。利用已获得的高剪接活性的S1和S11型断裂蛋白质内含子Ssp GyrB,分别将其参与剪接反应的首位半胱氨酸或末位天冬酰胺突变为丙氨酸,构建能够发生一端断裂的断裂蛋白质内含子。研究结果表明,突变后断裂蛋白质内含子的剪接反应几乎不发生,其断裂活性有不同程度的提高,获得了在大肠杆菌体内具有较高效断裂活性的断裂蛋白质内含子。这将为进一步研究其体外可控性剪接、构建高效的蛋白纯化系统和深入研究蛋白质内含子的剪接机制提供基础。     

蛋白质内含子的研究进展

自1990年发现第一个蛋白质内含子以来,对其研究愈加引起注意。蛋白质内含子是蛋白质剪接元件,可从前体蛋白中切除并将两侧外显子连接起来成为成熟蛋白质,标准的蛋白质剪接主要包括四步亲核置换反应,新近又发现一种反式剪接机制。蛋白质内含子的演化形成存在先天遗传和后天插入两种方式,但目前还没有直接的实验证据。数据库显示,蛋白质内含子有10个保守模体：A、N2、B、N4、C、D、E、H、F和G,它们在蛋白质剪接过程中具有不同的作用。作为蛋白质剪切元件的蛋白质内含子,是蛋白质工程中一个功能强大的工具,具有重要的实践意义。现对蛋白质内含子的命名、分布、结构、剪接方式以及应用前景等作一全面的综述。     

家蚕吡哆醛激酶cDNA的克隆、表达和基因结构分析

吡哆醛激酶（pyridoxal kinase,PLK, EC2.7.1.35）是维生素B₆关键代谢酶,其cDNA的克隆在昆虫类还未见报道。利用生物信息学原理和使用PCR方法,克隆出编码家蚕Bombyx mori吡哆醛激酶的cDNA (GenBank登录号DQ452397),体外原核表达成功,并对表达粗提产物进行了酶活检测。克隆到的cDNA含有一894 bp的完整可读框,编码一条分子量为33.1 kD,含298个氨基酸残基的蛋白质。序列比对显示此蛋白质与人类吡哆醛激酶具有52.84%的同一性,包含吡哆醛激酶家族共有的特征保守序列,但比哺乳动物和植物克隆到的吡哆醛激酶均少10多个氨基酸残基,几个有关键功能且在哺乳动物和植物中均保守的氨基酸残基在此蛋白中被替换。依据家蚕基因组数据库信息和PLK的cDNA,家蚕PLK基因包含5个外显子和4个内含子,跨越10 kb DNA序列,所有外显子/内含子交接点都遵从gt/ag剪接规则,基因的5′端启动子调控区发现有TATA-box和CAAT-box保守基序。     

内含子Ⅱ的作用机制和演化

内含子是广泛存在于原核生物的叶绿体、线粒体以及真核生物基因组中的非编码DNA序列,但其含有的调控元件却常常影响基因的表达效率。内含子的自我剪接在mRNA的成熟过程中起着重要作用,内含子也可利用逆转录剪接作用插入到同源或异源DNA中,同时也常常通过“外显子改组”来促进基因进化。文章重点介绍了近年来在内含子Ⅱ作用机制及演化方面研究的最新进展。     

Control of protein splicing by intein fragment reassembly.

Inteins are protein splicing elements that mediate their excision from precursor proteins and the joining of the flanking protein sequences (exteins). In this study, protein splicing was controlled by splitting precursor proteins within the Psp Pol-1 intein and expressing the resultant fragments in separate hosts. Reconstitution of an active intein was achieved by in vitro assembly of precursor fragments. Both splicing and intein endonuclease activity were restored. Complementary fragments from two of the three fragmentation positions tested were able to splice in vitro. Fragments resulting in redundant overlaps of intein sequences or containing affinity tags at the fragmentation sites were able to splice. Fragment pairs resulting in a gap in the intein sequence failed to splice or cleave. However, similar deletions in unfragmented precursors also failed to splice or cleave. Single splice junction cleavage was not observed with single fragments. In vitro splicing of intein fragments under native conditions was achieved using mini exteins. Trans-splicing allows differential modification of defined regions of a protein prior to extein ligation, generating partially labeled proteins for NMR analysis or enabling the study of the effects of any type of protein modification on a limited region of a protein.     

Faster Protein Splicing with the Nostoc punctiforme DnaE Intein Using Non-native Extein Residues

Inteins are naturally occurring intervening sequences that catalyze a protein splicing reaction resulting in intein excision and concatenation of the flanking polypeptides (exteins) with a native peptide bond. Inteins display a diversity of catalytic mechanisms within a highly conserved fold that is shared with hedgehog autoprocessing proteins. The unusual chemistry of inteins has afforded powerful biotechnology tools for controlling enzyme function upon splicing and allowing peptides of different origins to be coupled in a specific, time-defined manner. The extein sequences immediately flanking the intein affect splicing and can be defined as the intein substrate. Because of the enormous potential complexity of all possible flanking sequences, studying intein substrate specificity has been difficult. Therefore, we developed a genetic selection for splicing-dependent kanamycin resistance with no significant bias when six amino acids that immediately flanked the intein insertion site were randomized. We applied this selection to examine the sequence space of residues flanking the Nostoc punctiforme Npu DnaE intein and found that this intein efficiently splices a much wider range of sequences than previously thought, with little N-extein specificity and only two important C-extein positions. The novel selected extein sequences were sufficient to promote splicing in three unrelated proteins, confirming the generalizable nature of the specificity data and defining new potential insertion sites for any target. Kinetic analysis showed splicing rates with the selected exteins that were as fast or faster than the native extein, refuting past assumptions that the naturally selected flanking extein sequences are optimal for splicing.     

Post-translational environmental switch of RadA activity by extein–intein interactions in protein splicing

Post-translational control based on an environmentally sensitive intervening intein sequence is described. Inteins are invasive genetic elements that self-splice at the protein level from the flanking host protein, the exteins. Here we show in Escherichia coli and in vitro that splicing of the RadA intein located in the ATPase domain of the hyperthermophilic archaeon Pyrococcus horikoshii is strongly regulated by the native exteins, which lock the intein in an inactive state. High temperature or solution conditions can unlock the intein for full activity, as can remote extein point mutations. Notably, this splicing trap occurs through interactions between distant residues in the native exteins and the intein, in three-dimensional space. The exteins might thereby serve as an environmental sensor, releasing the intein for full activity only at optimal growth conditions for the native organism, while sparing ATP consumption under conditions of cold-shock. This partnership between the intein and its exteins, which implies coevolution of the parasitic intein and its host protein may provide a novel means of post-translational control.     

断裂蛋白质内含子的剪接机制、起源和进化

蛋白质内含子(intein)是具有自我催化活性的蛋白质. 翻译后,通过蛋白质剪接从蛋白质前体中去掉,并以肽键连接两侧蛋白质外显子(extein)形成成熟蛋白质. 断裂蛋白质内含子(split intein)在蛋白质内含子中部区域特定位点发生断裂,形成N端片段和C端片段,分别由基因组上相距较远的两个基因编码. 现在已知,它仅分布于蓝细菌和古细菌中. 断裂蛋白质内含子的N端片段和C端片段通过非共价键(如静电作用)相互识别,重建催化活性中心,介导蛋白质反式剪接. 断裂蛋白质内含子的发现进一步深化了人们对基因表达和蛋白质翻译后成熟过程复杂性的认识,而且它在蛋白质工程、蛋白质药物开发和蛋白质结构与功能研究等方面有非常广泛的应用. 本文试图综述断裂蛋白质内含子的分布、结构特征和剪接机制,并分析其可能的起源和进化途径.     

内含肽介导的生物学效应及其应用

蛋白质翻译产物在成熟过程中剪切释放出来的一段氨基酸序列称为“intein”---即内含肽。它与前体蛋白以框内融合的形式共同翻译,并内嵌于前体蛋白序列中。内含肽的解离以及内含肽两侧氨基酸序列的连接是在内含肽自身催化作用下完成的。本文将从内含肽的发现、结构特征和作用机理等方面对这种具有特殊意义的蛋白质成熟机制进行较为全面的论述,同时介绍了近年来发展起来的以内含肽介导的蛋白质剪接为基础的蛋白质纯化和改造技术。     

Inteins, valuable genetic elements in molecular biology and biotechnology

Inteins are internal protein elements that self-excise from their host protein and catalyze ligation of the flanking sequences (exteins) with a peptide bond. They are found in organisms in all three domains of life, and in viral proteins. Intein excision is a posttranslational process that does not require auxiliary enzymes or cofactors. This self-excision process is called protein splicing, by analogy to the splicing of RNA introns from pre-mRNA. Protein splicing involves only four intramolecular reactions, and a small number of key catalytic residues in the intein and exteins. Protein-splicing can also occur in trans. In this case, the intein is separated into N- and C-terminal domains, which are synthesized as separate components, each joined to an extein. The intein domains reassemble and link the joined exteins into a single functional protein. Understanding the cis- and trans-protein splicing mechanisms led to the development of intein-mediated protein-engineering applications, such as protein purification, ligation, cyclization, and selenoprotein production. This review summarizes the catalytic activities and structures of inteins, and focuses on the advantages of some recent intein applications in molecular biology and biotechnology.     

NMR and crystal structures of the Pyrococcus horikoshii RadA intein guide a strategy for engineering a highly efficient and promiscuous intein

In protein splicing, an intervening protein sequence (intein) in the host protein excises itself out and ligates two split host protein sequences (exteins) to produce a mature host protein. Inteins require the involvement for the splicing of the first residue of the extein that follows the intein (which is Cys, Ser, or Thr). Other extein residues near the splicing junctions could modulate splicing efficiency even when they are not directly involved in catalysis. Mutual interdependence between this molecular parasite (intein) and its host protein (exteins) is not beneficial for intein spread but could be advantageous for intein survival during evolution. Elucidating extein-intein dependency has increasingly become important since inteins are recognized as useful biotechnological tools for protein ligation. We determined the structures of one of inteins with high splicing efficiency, the RadA intein from Pyrococcus horikoshii (PhoRadA). The solution NMR structure and the crystal structures elucidated the structural basis for its high efficiency and directed our efforts of engineering that led to rational design of a functional minimized RadA intein. The crystal structure of the minimized RadA intein also revealed the precise interactions between N-extein and the intein. We systematically analyzed the effects at the -1 position of N-extein and were able to significantly improve the splicing efficiency of a less robust splicing variant by eliminating the unfavorable extein-intein interactions observed in the structure. This work provides an example of how unveiling structure-function relationships of inteins offer a promising way of improving their properties as better tools for protein engineering.     

Trans-splicing of an artificially split fungal mini-intein

Inteins are internal protein domains found inside the coding region of different proteins. They can autocatalytically self-excise from their host protein and ligate the protein flanks, called exteins, with a peptide bond via a post-translational process called protein cis-splicing. In contrast, protein trans-splicing involves inteins split into an N- and a C-terminal domain. Both domains are synthesized as two separate components and each joined to an extein; the intein domains can reassemble and link the joined exteins into one functional protein. In this study, we introduced three split sites into the PRP8 mini-intein of Penicillium chrysogenum and demonstrated for the first time trans-splicing of a fungal PRP8 intein. Two of the sites introduced allowed splicing to occur in trans while the third was not functional.