Recent Technologies for Genetic Code Expansion and their Implications on Synthetic Biology Applications

Genetic code expansion (GCE) enables the site-specific incorporation of non-canonical amino acids as novel building blocks for the investigation and manipulation of proteins. The advancement of genetic code expansion has been benefited from the development of synthetic biology, while genetic code expansion also helps to create more synthetic biology tools. In this review, we summarize recent advances in genetic code expansion brought by synthetic biology progresses, including engineering of the translation machinery, genome-wide codon reassignment, and the biosynthesis of non-canonical amino acids. We highlight the emerging application of this technology in construction of new synthetic biology parts, circuits, chassis, and products.     

Detecting oxidative post-translational modifications in proteins

Summary. Oxidative stress induces various post-translational modifications (PTM); some are reversible in vivo via enzymatic catalysis. The present paper reviews specific procedures for the detection of oxidative PTM in proteins, most of them including electrophoresis. Main topics are carbonylated and glutathionylated proteins as well as modification of selected amino acids (Cys, Tyr, Met, Trp, Lys).     

非自然氨基酸在蛋白质中的引入方法及其在化学生物学中的应Q用

蛋白质是生命的物质基础,在生物体中行驶着极为重要的功能,各种细胞活动和生命过程的发生都需要蛋白质的参与。例如,DNA复制转录,RNA翻译,以及信号传导等过程中发挥关键作用的聚合酶、翻译复合物、信号传导受体等都是蛋白质。正常细胞体内的所有蛋白质都由20种天然存在的氨基酸组成,它们通过立体构象变化及翻译后修饰等来控制其功能的发挥。而在实际应用中,学者们为了实现不同的研究目的,发展了很多在蛋白质中引入20种自然氨基酸之外的非自然氨基酸(unnatural amino acid,UAA)的方法,如化学修饰合成、体外翻译、遗传密码扩展等,从而将蛋白质的性质根据研究和应用的需要进行拓展。本文综述了各类化学与生物中引入非自然氨基酸的方法,并介绍了非自然氨基酸在化学生物学研究中的最新应用。     

Increasing the chemical space of proteins in living cells via genetic code expansion

In recent years it has become possible to genetically encode an expanded set of designer amino acids with tailored chemical and physical properties (dubbed unnatural amino acids, UAAs) into proteins in living cells by expanding the genetic code. Together with developments in chemistries that are amenable to and selective within physiological settings, these strategies have started to have a big impact on biological studies, as they enable exciting in cellulo applications. Here we highlight recent advances to covalently stabilize transient protein–protein interactions and capture enzyme substrate-complexes in living cells using proximity-triggered and residue-selective photo-induced crosslinking approaches. Furthermore, we describe recent efforts in controlling enzyme activity with photocaged UAAs and in extending their application to a variety of enzymatic scaffolds. In addition, we discuss the site-specific incorporation of UAAs mimicking post-translational modifications (PTMs) and approaches to generate natively-linked ubiquitin–protein conjugates to probe the role of PTMs in modulating complex cellular networks.     

Techniques for studying protein heterogeneity and post-translational modifications

Proteins often undergo several post-translational modification steps in parallel to protein folding. These modifications can be transient or of a more permanent nature. Most modifications are, however, susceptible to alteration during the lifespan of proteins. Post-translational modifications thus generate variability in proteins that are far beyond that provided by the genetic code. Co- and post-translational modifications can convert the 20 specific codon-encoded amino acids into more than 100 variant amino acids with new properties. These, and a number of other modifications, can considerably increase the information content and functional repertoire of proteins, thus making their analysis of paramount importance for diagnostic and basic research purposes. Various methods used in proteomics, such as 2D gel electrophoresis, 2D liquid chromatography, mass spectrometry, affinity-based analytical methods, interaction analyses, ligand blotting techniques, protein crystallography and structure–function predictions, are all applicable for the analysis of these numerous secondary modifications. In this review, examples of some of these techniques in studying the heterogeneity of proteins are highlighted. In the future, these methods will become increasingly useful in biomarker searches and in clinical diagnostics.     

Techniques for studying protein heterogeneity and post-translational modifications

Proteins often undergo several post-translational modification steps in parallel to protein folding. These modifications can be transient or of a more permanent nature. Most modifications are, however, susceptible to alteration during the lifespan of proteins. Post-translational modifications thus generate variability in proteins that are far beyond that provided by the genetic code. Co- and post-translational modifications can convert the 20 specific codon-encoded amino acids into more than 100 variant amino acids with new properties. These, and a number of other modifications, can considerably increase the information content and functional repertoire of proteins, thus making their analysis of paramount importance for diagnostic and basic research purposes. Various methods used in proteomics, such as 2D gel electrophoresis, 2D liquid chromatography, mass spectrometry, affinity-based analytical methods, interaction analyses, ligand blotting techniques, protein crystallography and structure-function predictions, are all applicable for the analysis of these numerous secondary modifications. In this review, examples of some of these techniques in studying the heterogeneity of proteins are highlighted. In the future, these methods will become increasingly useful in biomarker searches and in clinical diagnostics.     

Bioconjugation of therapeutic proteins and enzymes using the expanded set of genetically encoded amino acids

The last decade has witnessed striking progress in the development of bioorthogonal reactions that are strictly directed towards intended sites in biomolecules while avoiding interference by a number of physical and chemical factors in biological environment. Efforts to exploit bioorthogonal reactions in protein conjugation have led to the evolution of protein translational machineries and the expansion of genetic codes that systematically incorporate a range of non-natural amino acids containing bioorthogonal groups into recombinant proteins in a site-specific manner. Chemoselective conjugation of proteins has begun to find valuable applications to previously inaccessible problems. In this review, we describe bioorthogonal reactions useful for protein conjugation, and biosynthetic methods that produce proteins amenable to those reactions through an expanded genetic code. We then provide key examples in which novel protein conjugates, generated by the genetic incorporation of a non-natural amino acid and the chemoselective reactions, address unmet needs in protein therapeutics and enzyme engineering.     

Reprogramming the amino-acid substrate specificity of orthogonal aminoacyl-tRNA synthetases to expand the genetic code of eukaryotic cells

The genetic code of living organisms has been expanded to allow the site-specific incorporation of unnatural amino acids into proteins in response to the amber stop codon UAG. Numerous amino acids have been incorporated including photo-crosslinkers, chemical handles, heavy atoms and post-translational modifications, and this has created new methods for studying biology and developing protein therapeutics and other biotechnological applications. Here we describe a protocol for reprogramming the amino-acid substrate specificity of aminoacyl-tRNA synthetase enzymes that are orthogonal in eukaryotic cells. The resulting aminoacyl-tRNA synthetases aminoacylate an amber suppressor tRNA with a desired unnatural amino acid, but no natural amino acids, in eukaryotic cells. To achieve this change of enzyme specificity, a library of orthogonal aminoacyl-tRNA synthetase is generated and genetic selections are performed on the library in Saccharomyces cerevisiae. The entire protocol, including characterization of the evolved aminoacyl-tRNA synthetase in S. cerevisiae, can be completed in approximately 1 month.     

Unremitting progresses for phosphoprotein synthesis

Phosphorylation, one of the important protein post-translational modifications, is involved in many essential cellular processes. Site-specifical and homogeneous phosphoproteins can be used as probes for elucidating the protein phosphorylation network and as potential therapeutics for interfering their involved biological events. However, the generation of phosphoproteins has been challenging owing to the limitation of chemical synthesis and protein expression systems. Despite the pioneering discoveries in phosphoprotein synthesis, over the past decade, great progresses in this field have also been made to promote the biofunctional exploration of protein phosphorylation largely. Therefore, in this review, we mainly summarize recent advances in phosphoprotein synthesis, which includes five sections: 1) synthesis of the nonhydrolyzable phosphorylated amino acid mimetic building blocks, 2) chemical total and semisynthesis strategy, 3) in-cell and in vitro genetic code expansion strategy, 4) the late-stage modification strategy, 5) nonoxygen phosphoprotein synthesis.     

Chemogenetic and optogenetic control of post-translational modifications through genetic code expansion

Post-translational modifications (PTMs) of proteins extensively diversify the biological information flow from the genome to the proteome and thus have profound pathophysiological implications. Precise dissection of the regulatory networks of PTMs benefits from the ability to achieve conditional control through external optogenetic or chemogenetic triggers. Genetic code expansion provides a unique solution by allowing for site-specific installation of functionally masked unnatural amino acids (UAAs) into proteins, such as enzymes and enzyme substrates, rendering them inert until rapid activation through exposure to light or small molecules. Here, we summarize the most recent advances harnessing this methodology to study various forms of PTMs, as well as generalizable approaches to externally control nodes-of-interest in PTM networks.     

MAPRes: an efficient method to analyze protein sequence around post-translational modification sites

Functional switches are often regulated by dynamic protein modifications. Assessing protein functions, in vivo, and their functional switches remains still a great challenge in this age of development. An alternative methodology based on in silico procedures may facilitate assessing the multifunctionality of proteins and, in addition, allow predicting functions of those proteins that exhibit their functionality through transitory modifications. Extensive research is ongoing to predict the sequence of protein modification sites and analyze their dynamic nature. This study reports the analysis performed on phosphorylation, Phospho.ELM (version 3.0) and glycosylation, OGlycBase (version 6.0) data for mining association patterns utilizing a newly developed algorithm, MAPRes. This method, MAPRes (Mining Association Patterns among preferred amino acid residues in the vicinity of amino acids targeted for post-translational modifications), is based on mining association among significantly preferred amino acids of neighboring sequence environment and modification sites themselves. Association patterns arrived at by association pattern/rule mining were in significant conformity with the results of different approaches. However, attempts to analyze substrate sequence environment of phosphorylation sites catalyzed for Tyr kinases and the sequence data for O-GlcNAc modification were not successful, due to the limited data available. Using the MAPRes algorithm for developing an association among PTM site with its vicinal amino acids is a valid method with many potential uses: this is indeed the first method ever to apply the association pattern mining technique to protein post-translational modification data.     

Using a quadruplet codon to expand the genetic code of an animal

Genetic code expansion in multicellular organisms is currently limited to the use of repurposed amber stop codons. Here, we introduce a system for the use of quadruplet codons to direct incorporation of non-canonical amino acids in vivo in an animal, the nematode worm Caenorhabditis elegans. We develop hybrid pyrrolysyl tRNA variants to incorporate non-canonical amino acids in response to the quadruplet codon UAGA. We demonstrate the efficiency of the quadruplet decoding system by incorporating photocaged amino acids into two proteins widely used as genetic tools. We use photocaged lysine to express photocaged Cre recombinase for the optical control of gene expression and photocaged cysteine to express photo-activatable caspase for light inducible cell ablation. Our approach will facilitate the routine adoption of quadruplet decoding for genetic code expansion in eukaryotic cells and multicellular organisms.     

基于古菌酪氨酰tRNA合成酶非天然氨基酸插入的研究进展

构筑蛋白质的编码信息存在于高度保守的密码子表中,而生物体仅利用20种天然氨基酸,就能排列组合出不同的蛋白质来行使多种生物学功能。通过合成生物学的飞速发展,使得在蛋白质合成中可控地引入非天然氨基酸成为可能。这极大地拓展了蛋白质的结构和功能,并为生物学工具的开发和生物生理过程的研究提供了便利。具有活性基团的非天然氨基酸可以广泛地应用于蛋白质结构研究、蛋白质功能调控以及新型生物材料构建和医药研发等诸多领域。基因密码子拓展技术利用正交翻译系统,通过重新分配密码子改造中心法则,可以在蛋白质的指定位点引入非天然氨基酸。系统地介绍了目前提升密码子拓展技术插入非天然氨基酸效率的方法,包括tRNA以及氨酰tRNA合成酶的各种突变方法和翻译辅助因子的改造。汇总了利用古细菌酪氨酰tRNA合成酶插入的非天然氨基酸和突变位点并总结了密码子拓展技术在生物医药领域的前沿进展。最后讨论了该项技术目前所面临的挑战,如可利用的密码子数量不多、正交翻译系统的种类有限和非天然氨基酸多插效率低下。希望能够帮助研究者建立适合的非天然氨基酸插入方法并推动密码子拓展技术进一步发展。     

Signature of a Primitive Genetic Code in Ancient Protein Lineages

The genetic code is the syntactic foundation underlying the structure and function of every protein in the history of the biological world. Its highly ordered degenerate complexity suggests an incremental evolution, the result of a combination of selective, mechanistic, and random processes. These evolutionary processes are still poorly understood and remain an open question in the study of early life on Earth. We perform a compositional analysis of ribosomal proteins and ATPase subunits in bacterial and archaeal lineages, using conserved positions that came and remained under purifying selection before and up to the most recent common ancestor. An observable shift in amino acid usage at these conserved positions likely provides an untapped window into the history of protein sequence space, allowing events of genetic code expansion to be identified. We identify Cys, Glu, Phe, Ile, Lys, Val, Trp, and Tyr as recent additions to the genetic code, with Asn, Gln, Gly, and Leu among the more ancient. Our observations are consistent with a scenario in which genetic code expansion primarily favored amino acids that promoted an increase in polypeptide size and functionality. We propose that this expansion would have been critical in the takeover of many RNA-mediated processes, as well as the addition of novel biological functions inaccessible to an RNA-based physiology, such as crossing lipid membranes. Thus, expansion of the genetic code likely set the stage for the transition from RNA-based to protein-based life. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Engineering Translation Components for Genetic Code Expansion

The expansion of the genetic code consisting of four bases and 20 amino acids into diverse building blocks has been an exciting topic in synthetic biology. Many biochemical components are involved in gene expression; therefore, adding a new component to the genetic code requires engineering many other components that interact with it. Genetic code expansion has advanced significantly for the last two decades with the engineering of several components involved in protein synthesis. These components include tRNA/aminoacyl-tRNA synthetase, new codons, ribosomes, and elongation factor Tu. In addition, biosynthesis and enhanced uptake of non-canonical amino acids have been attempted and have made meaningful progress. This review discusses the efforts to engineer these translation components, to improve the genetic code expansion technology.     

Reassignment of sense codons in vivo

The genetic code maps one or more of the 61 sense codons onto a set of 20 canonical amino acids. Reassignment of sense codons to non-canonical amino acids in model organisms such as Escherichia coli has been achieved through manipulation of the cellular protein synthesis machinery. Specifically, control of amino acid pools, coupled with engineering of the aminoacyl-tRNA synthetase activity of the host, has enabled assignment of sense codons to a wide variety of non-canonical amino acids under conditions routinely used for expression of recombinant proteins. Codon reassignment is leading to important advances in protein engineering and bioorganic chemistry. Here we summarize some of those advances, and provide detailed protocols for codon reassignment.     

Visualizing the complexity of proteins in living cells with genetic code expansion

Genetic code expansion has emerged as an enabling tool to provide insight into functions of understudied proteinogenic species, such as small proteins and peptides, and to probe protein biophysics in the cellular context. Here, we discuss recent technical advances and applications of genetic code expansion in cellular imaging of complex mammalian protein species, along with considerations and challenges on using the method.     

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。     

Modern and prebiotic amino acids support distinct structural profiles in proteins

The earliest proteins had to rely on amino acids available on early Earth before the biosynthetic pathways for more complex amino acids evolved. In extant proteins, a significant fraction of the ‘late’ amino acids (such as Arg, Lys, His, Cys, Trp and Tyr) belong to essential catalytic and structure-stabilizing residues. How (or if) early proteins could sustain an early biosphere has been a major puzzle. Here, we analysed two combinatorial protein libraries representing proxies of the available sequence space at two different evolutionary stages. The first is composed of the entire alphabet of 20 amino acids while the second one consists of only 10 residues (ASDGLIPTEV) representing a consensus view of plausibly available amino acids through prebiotic chemistry. We show that compact conformations resistant to proteolysis are surprisingly similarly abundant in both libraries. In addition, the early alphabet proteins are inherently more soluble and refoldable, independent of the general Hsp70 chaperone activity. By contrast, chaperones significantly increase the otherwise poor solubility of the modern alphabet proteins suggesting their coevolution with the amino acid repertoire. Our work indicates that while both early and modern amino acids are predisposed to supporting protein structure, they do so with different biophysical properties and via different mechanisms.     

Ubiquitin modifications

Protein ubiquitination is a dynamic multifaceted post-translational modification involved in nearly all aspects of eukaryotic biology. Once attached to a substrate, the 76-amino acid protein ubiquitin is subjected to further modifications, creating a multitude of distinct signals with distinct cellular outcomes, referred to as the ''ubiquitin code''. Ubiquitin can be ubiquitinated on seven lysine (Lys) residues or on the N-terminus, leading to polyubiquitin chains that can encompass complex topologies. Alternatively or in addition, ubiquitin Lys residues can be modified by ubiquitin-like molecules (such as SUMO or NEDD8). Finally, ubiquitin can also be acetylated on Lys, or phosphorylated on Ser, Thr or Tyr residues, and each modification has the potential to dramatically alter the signaling outcome. While the number of distinctly modified ubiquitin species in cells is mind-boggling, much progress has been made to characterize the roles of distinct ubiquitin modifications, and many enzymes and receptors have been identified that create, recognize or remove these ubiquitin modifications. We here provide an overview of the various ubiquitin modifications present in cells, and highlight recent progress on ubiquitin chain biology. We then discuss the recent findings in the field of ubiquitin acetylation and phosphorylation, with a focus on Ser65-phosphorylation and its role in mitophagy and Parkin activation.