非天然氨基酸正交翻译技术：一种新型基因工程活病毒疫苗研发技术

非天然氨基酸正交翻译技术利用外源的非天然氨基酸氨酰tRNA合成酶(aaRS)基因和对应的tRNA基因构建非天然氨基酸正交翻译系统(Orthogonal translation system)。该正交翻译系统能利用终止密码子在蛋白翻译过程中将非天然氨基酸定点插入目标多肽链中。该技术不但是一种新的蛋白质生化研究工具,在新型基因工程病毒疫苗研究中更具有划时代的意义。利用人为构建的具有非天然氨基酸正交翻译系统的转基因细胞,通过在病毒复制的关键基因中引入提前终止密码子构建的突变病毒,在添加非天然氨基酸的情况下该基因仍能完整表达从而完成病毒的复制和传代,但该突变病毒在正常细胞(无非天然氨基酸正交翻译系统的宿主细胞)中因复制关键基因不能完整表达而无法复制传代,因而是一种复制缺陷型病毒。这种复制缺陷型病毒用作疫苗时兼具了减毒活疫苗免疫效果良好与灭活疫苗安全性高的优点,是一种较为理想的活病毒疫苗。文中简要综述了非天然氨基酸正交翻译技术在新型复制缺陷活病毒疫苗研究中的应用及其前景。     

微生物tRNA与密码子系统应用研究进展*

tRNA作为生命中心法则中翻译过程的重要参与分子,其种类、丰度都会对蛋白质的正常合成产生巨大影响。近年来通过对微生物tRNA的结构功能以及合成修饰过程的解析获得诸多启发,开展密码子扩展的研究,实现将非天然氨基酸引入特定位置从而获得新功能蛋白。同时,通过化学合成微生物基因组开展的密码子重编码工作将释放更多的密码子与tRNA用于更加广泛的密码子扩展研究。对微生物tRNA与密码子系统在合成生物学中的最新应用研究进展进行了综述,并讨论其未来的发展趋势。     

氨酰-tRNA合成酶的研究进展

氨酰-tRNA合成酶催化特异的氨基酸与同源tRNA氨酰化,从而保证了遗传密码翻译的忠实性。这些古老而保守的蛋白质分子除了具有酶的功能外,在哺乳动物细胞中还发现了多种其他功能,具有重要的应用价值。在寻找具有全新作用机制的新抗生素以应对日益严重的抗生素耐药现象过程中,氨酰-tRNA合成酶是细菌蛋白质合成过程中重要的、新颖的靶标,成为关注的重点。定向突变的氨酰-tRNA合成酶可以用来定点掺入非天然氨基酸,扩展蛋白质工程。今后,随着人们对氨酰-tRNA合成酶研究的不断深入,它们还可能用来治疗肿瘤等多种疾病。     

氨酰tRNA合成酶的分子网络和功能

氨酰tRNA合成酶是生命进化过程中最早出现的一类蛋白质,氨酰tRNA合成酶帮助氨基酸转移到相应的tRNA上,进而参与蛋白质的合成保证了生命体的严谨性和多样性.随着后基因组时代的到来,氨酰tRNA合成酶的结构和功能成为新的研究热点.结构生物学和生物信息学的研究结果表明,氨酰tRNA合成酶在真核生物体内以多聚复合物的形式行使功能,形成复杂的分子网络体系.最新的实验证据显示,氨酰tRNA合成酶不但是蛋白质合成过程中一类最重要的酶,而且参与了转录、翻译水平的调控、RNA剪接、信号传导和免疫应答等众多生命活动.     

合成生物学中的正交遗传系统

并行、独立的正交系统是合成生物学的重要研究基础之一,这个系统与自然界的生物系统及其组成交叉很少或没有交叉。它的组成包括非天然碱基对、移位密码子、非天然氨基酸、正交的氨酰tRNA合成酶、RNA聚合酶和启动子、正交核糖体等。这些正交系统的组成部分可以一起组成系统发挥作用,也可以各自单独在生物体系中应用,它们给生物带来新的特性,也为研究人员提供了新的生物学研究方法。     

tRNA介导蛋白质工程

在遗传信息从DNA到蛋白质流动的过程中,tRNA携带特异的氨基酸参与蛋白质合成,对于维持蛋白质翻译的忠实性起着非常重要的作用。生物体内共有20种氨酰tRNA合成酶,每一种均对应于一种氨基酸和一个tRNA类型。但是这种翻译过程仅仅限于20种天然氨基酸,因此在进行传统的蛋白质工程研究时常常受到限制。事实上,在蛋白质工程中借助于校正tRNA定点掺入非天然氨基酸可以提供蛋白质的结构信息,改进蛋白质检测与分离的方法,甚至赋予蛋白质某些新的特性。随着生物技术的发展和完善,tRNA介导蛋白质工程将不仅在蛋白质工程中发挥潜能,而且在研制新型生物材料和疾病诊断及药物治疗方面起到推动作用。     

tRNA介导蛋白质工程

在遗传信息从DNA到蛋白质流动的过程中,tRNA携带特异的氨基酸参与蛋白质合成,对于维持蛋白质翻译的忠实性起着非常重要的作用,生物体内共有20种氨酰tRNA合成酶,每一种均对尖于一种氨基酸和一个tRNA类型,但是这种翻译过程仅仅限于20种天然氨基酸,因此在进行传统的蛋白质工程研究时常常受到限制,事实上,在蛋白质工程中借助于校正 tRNA定点掺入非天然氨基酸可以提供蛋白质的结构信息,改进蛋白质检测与分离的方法,甚至赋予蛋白质某些新的特性,随着生物技术的发展和完善,tRNA介导蛋白质工程将不仅在蛋白质工程中发挥潜能,而且在研制新型生物材料和疾病诊断及药物治疗方面起到推动作用。     

氨酰tRNA合成酶作用机制及其应用

氨酰tRNA合成酶（aminoacyl tRNA synthetases, aaRSs）通过催化氨基酸与相应tRNA的氨酰化以保证遗传信息翻译的准确性,在生物体内具有重要作用。近年来,随着对aaRS催化机制理解的不断加深,aaRS的应用逐渐成为研究热点。在细菌中,aaRS活性被抑制后会导致其生命活动发生紊乱,根据aaRS在人体与病原菌内不同的催化特点设计针对病原体的特异性aaRS抑制剂,将有助于开发以aaRS为靶标的新型抗生素。另外,通过突变aaRS可以在蛋白质序列中定点掺入非天然氨基酸,扩展蛋白质工程。本文简述了aaRS的分类、结构与功能的特点,并在此基础上综述了aaRS在研发新型抑制剂,设计改造特殊蛋白质等方面的应用。     

无细胞体系非天然蛋白质合成研究进展

无细胞非天然蛋白质合成作为蛋白质研究的新兴手段,已成功用于表征蛋白质分子间、蛋白质与核酸分子间相互作用等基础科学研究及医药蛋白、蛋白质材料等工业生产领域。无细胞非天然蛋白质合成系统不需维持细胞的生长,无细胞膜阻碍,可依据研究目的添加基因元件或化学物质从而增强工程设计和过程调控的自由性;也可赋予蛋白质新的特性、结构及功能,如可实现蛋白翻译后修饰、反应手柄引入、生物物理探针及多聚蛋白质合成等。文中系统地综述了目前应用于无细胞蛋白质合成系统中的非天然氨基酸嵌入方法,包括全局抑制及基于正交翻译体系的终止密码子抑制、移码抑制、有义密码子再分配和非天然碱基等方法的研究进展,及非天然氨基酸在蛋白质修饰、生物物理探针、酶工程、蛋白质材料以及医药蛋白质生产等领域的应用进展,并分析了该体系的发展前景及广泛工业化应用的机遇与挑战。     

氨酰tRNA合成酶作用机制及其应用北大核心CSCD

氨酰tRNA合成酶(aminoacyl-tRNA synthetases,aaRSs)通过催化氨基酸与相应tRNA的氨酰化以保证遗传信息翻译的准确性,在生物体内具有重要作用。近年来,随着对aaRS催化机制理解的不断加深,aaRS的应用逐渐成为研究热点。在细菌中,aaRS活性被抑制后会导致其生命活动发生紊乱,根据aaRS在人体与病原菌内不同的催化特点设计针对病原体的特异性aaRS抑制剂,将有助于开发以aaRS为靶标的新型抗生素。另外,通过突变aaRS可以在蛋白质序列中定点掺入非天然氨基酸,扩展蛋白质工程。本文简述了aaRS的分类、结构与功能的特点,并在此基础上综述了aaRS在研发新型抑制剂,设计改造特殊蛋白质等方面的应用。     

A chemical toolkit for proteins--an expanded genetic code

Recently, a method to encode unnatural amino acids with diverse physicochemical and biological properties genetically in bacteria, yeast and mammalian cells was developed. Over 30 unnatural amino acids have been co-translationally incorporated into proteins with high fidelity and efficiency using a unique codon and corresponding transfer-RNA:aminoacyl-tRNA-synthetase pair. This provides a powerful tool for exploring protein structure and function in vitro and in vivo, and for generating proteins with new or enhanced properties.     

Evolved orthogonal ribosomes enhance the efficiency of synthetic genetic code expansion

In vivo incorporation of unnatural amino acids by amber codon suppression is limited by release factor-1-mediated peptide chain termination. Orthogonal ribosome-mRNA pairs function in parallel with, but independent of, natural ribosomes and mRNAs. Here we show that an evolved orthogonal ribosome (ribo-X) improves tRNA(CUA)-dependent decoding of amber codons placed in orthogonal mRNA. By combining ribo-X, orthogonal mRNAs and orthogonal aminoacyl-tRNA synthetase/tRNA pairs in Escherichia coli, we increase the efficiency of site-specific unnatural amino acid incorporation from approximately 20% to >60% on a single amber codon and from <1% to >20% on two amber codons. We hypothesize that these increases result from a decreased functional interaction of the orthogonal ribosome with release factor-1. This technology should minimize the functional and phenotypic effects of truncated proteins in experiments that use unnatural amino acid incorporation to probe protein function in vivo.     

Site-specific labeling of proteins with NMR-active unnatural amino acids

A large number of amino acids other than the canonical amino acids can now be easily incorporated in vivo into proteins at genetically encoded positions. The technology requires an orthogonal tRNA/aminoacyl-tRNA synthetase pair specific for the unnatural amino acid that is added to the media while a TAG amber or frame shift codon specifies the incorporation site in the protein to be studied. These unnatural amino acids can be isotopically labeled and provide unique opportunities for site-specific labeling of proteins for NMR studies. In this perspective, we discuss these opportunities including new photocaged unnatural amino acids, outline usage of metal chelating and spin-labeled unnatural amino acids and expand the approach to in-cell NMR experiments.     

Adding amino acids to the genetic repertoire

Considerable progress has been made in expanding the number and nature of genetically encoded amino acids in Escherichia coli, yeast and mammalian cells in the past four years. To date, over 30 unnatural amino acids have been cotranslationally incorporated into proteins with high fidelity and efficiency by means of a unique codon and corresponding orthogonal tRNA-aminoacyl-tRNA synthetase pair. The incorporated amino acids contain spectroscopic probes, post-translational modifications, metal chelators, photoaffinity labels and unique functional groups. The ability to genetically encode additional amino acids, beyond the common 20, provides a powerful approach for probing protein structure and function both in vitro and in vivo, as well as generating proteins with new or enhanced properties.     

An expanding genetic code

A general method was recently developed that makes it possible to genetically encode unnatural amino acids (UAAs) with diverse physical, chemical or biological properties in Escherichia coli, yeast, and mammalian cells. Over 30 UAAs have been cotranslationally incorporated into proteins with high fidelity and efficiency by means of a unique codon and corresponding tRNA-synthetase pair. A key feature of this methodology is the orthogonality between the new translational components and their endogenous host counterparts. Specifically, the codon for the UAA should not encode a common amino acid; neither the new tRNA nor cognate aminoacyl tRNA synthetase should cross-react with any endogenous tRNA-synthetase pairs; and the new synthetase should recognize only the UAA and not any of the 20 common amino acids. This methodology provides a powerful tool for exploring protein structure and function both in vitro and in vivo, as well as generating proteins with new or enhanced properties.     

The pyrrolysine translational machinery as a genetic-code expansion tool

The discovery of pyrrolysine not only expanded the set of the known proteinogenic amino acids but also revealed unusual features of its encoding mechanism. The engagement of a canonical stop codon and a unique aminoacyl-tRNA synthetase-tRNA pair that can be used to accommodate a broad range of unnatural amino acids while maintaining strict orthogonality in a variety of prokaryotic and eukaryotic expression systems has proven an invaluable combination. Within a few years since its properties were elucidated, the pyrrolysine translational machinery has become a popular choice for the synthesis of recombinant proteins bearing a wide variety of otherwise hard-to-introduce functional groups. It is also central to the development of new synthetic strategies that rely on stop-codon suppression.     

Introduction of specialty functions by the position-specific incorporation of nonnatural amino acids into proteins through four-base codon/anticodon pairs

Position-specific incorporation of nonnatural amino acids into proteins (nonnatural mutagenesis) via an in vitro protein synthesizing system was applied to incorporate a variety of amino acids carrying specialty side groups. A list of nonnatural amino acids thus far successfully incorporated through in vitro translation systems is presented. The position of nonnatural amino acid incorporation was directed by four-base codon/anticodon pairs such as CGGG/CCCG and AGGU/ACCU. The four-base codon strategy was more efficient than the amber codon strategy and could incorporate multiple nonnatural amino acids into single proteins. This multiple mutagenesis will find wide applications, especially in building paths of electron transfer on proteins. The extension of translation systems by the introduction of nonnatural amino acids, four-base codon/anticodon pairs, orthogonal tRNAs, and artificial aminoacyl tRNA synthetases, is a promising approach towards the creation of "synthetic microorganisms" with specialty functions.     

Genetic incorporation of unnatural amino acids into proteins in mammalian cells

We developed a general approach that allows unnatural amino acids with diverse physicochemical and biological properties to be genetically encoded in mammalian cells. A mutant Escherichia coli aminoacyl-tRNA synthetase (aaRS) is first evolved in yeast to selectively aminoacylate its tRNA with the unnatural amino acid of interest. This mutant aaRS together with an amber suppressor tRNA from Bacillus stearothermophilus is then used to site-specifically incorporate the unnatural amino acid into a protein in mammalian cells in response to an amber nonsense codon. We independently incorporated six unnatural amino acids into GFP expressed in CHO cells with efficiencies up to 1 mug protein per 2 x 10(7) cells; mass spectrometry confirmed a high translational fidelity for the unnatural amino acid. This methodology should facilitate the introduction of biological probes into proteins for cellular studies and may ultimately facilitate the synthesis of therapeutic proteins containing unnatural amino acids in mammalian cells.     

In vitro selection for sense codon suppression

The universal genetic code links the 20 naturally occurring amino acids to the 61 sense codons. Previously, the UAG amber stop codon (a nonsense codon) has been used as a blank in the code to insert natural and unnatural amino acids via nonsense suppression. We have developed a selection methodology to investigate whether the unnatural amino acid biocytin could be incorporated into an mRNA display library at sense codons. In these experiments we probed a single randomized NNN codon with a library of 16 orthogonal, biocytin-acylated tRNAs. In vitro selection for efficient incorporation of the unnatural amino acid resulted in templates containing the GUA codon at the randomized position. This sense suppression occurs via Watson-Crick pairing with similar efficiency to UAG-mediated nonsense suppression. These experiments suggest that sense codon suppression is a viable means to expand the chemical and functional diversity of the genetic code.