Sense codon-dependent introduction of unnatural amino acids into multiple sites of a protein

Cell-free protein synthesis, driven by a crude S30 extract from Escherichia coli, has been applied to the preparation of proteins containing unnatural amino acids at specific positions. We have developed methods for inactivating tRNA(Asp) and tRNA(Phe) within a crude E. coli tRNA by an antisense treatment and for digesting most of the tRNA within the S30 extract without essential damage to the ribosomal activity. In the present study, we applied these methods to the substitution of Asp and Phe residues of the HIV-1 protease with unnatural amino acids. With 10 mM Mg(2+), the translation efficiency was higher than that with the other tested concentration, and the misreading efficiency was low. The protease mRNA was translated in the presence of an antisense DNA-treated tRNA mixture and 2-naphthylalanyl- and/or p-phenylazophenylalanyl-tRNA. The results suggest that a good portion of the translation products are substituted at all of the seven positions originally occupied by Asp or Phe.     

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。     

Automatic Edman microsequencing of peptides containing multiple unnatural amino acids

It is now routine using automatic Edman microsequencing to determine the primary structure of peptides or proteins containing natural amino acids; however, a deficiency in the ability to readily sequence peptides containing unnatural amino acids remains. With the advent of synthetic peptide chemistry, combinatorial chemistry, and the large number of commercially available unnatural amino acids, there is a need for efficient and accurate structure determination of short peptides containing many unnatural amino acids. In this study, 35 commercially available alpha-unnatural amino acids were selected to determine their elution profile on an ABI protein sequencer. Using a slightly modified gradient program, 19 of these 35 PTH amino acids can be readily resolved and distinguished from common PTH amino acids at low picomole levels. These unnatural amino acids in conjunction with the 20 natural amino acids can be used as building blocks to construct peptide libraries, and peptide beads isolated from these libraries can be readily microsequenced. To demonstrate this, we synthesized a simple tripeptide "one-bead one-compound" combinatorial library containing 14 unnatural and 19 natural amino acids and screened this library for streptavidin-binding ligands. Microsequencing of the isolated peptide-beads revealed the novel motif Bpa-Phe(4-X)-Aib, wherein X = H, OH, and CH3.     

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.     

Asymmetric synthesis of L-homophenylalanine by equilibrium-shift using recombinant aromatic L-amino acid transaminase

L-Homophenylalanine (L-HPA) was asymmetrically synthesized from 2-oxo-4-phenylbutyric acid (2-OPBA) and L-aspartate using a recombinant aromatic amino acid transaminase (AroAT). To screen microorganisms having such an L-specific AroAT with a relaxed substrate inhibition in the asymmetric synthesis of unnatural amino acids, enrichment cultures were performed in a minimal media containing 50 mM L-HPA as a sole nitrogen source. To reduce the intracellular background synthetic activity by amino acid pools in the cells, a two-step screening method was used. The putative AroAT (i.e., AroATEs) from the screened Enterobacter sp. BK2K-1 was cloned, sequenced, and overexpressed in E. coli cells. The activity of the overexpressed AroATEs was 314-fold higher than that of the wild-type cell. The substrate specificities of the enzyme and homology search revealed that the cloned transaminase is true AroAT. The AroATEs showed a substrate inhibition by 2-OPBA from 40 mM in the asymmetric synthesis, which made it difficult to perform batch asymmetric synthesis of L-HPA at high concentrations of 2-OPBA. To avoid the substrate inhibition by 2-OPBA, intermittent addition of the solid-state substrate was attempted to obtain a high concentration of L-HPA. By using the cell extract (75 U) obtained from the recombinant E. coli harboring the AroATEs gene, the asymmetric synthesis of L-HPA at 840 mM of 2-OPBA resulted in >94% of conversion yield and >99% ee of L-HPA of optical purity. Due to the low solubility (<2 mM) of L-HPA in the reaction buffer, synthesized L-HPA was continuously precipitated in the reaction media, which drives the reaction equilibrium towards the product formation. After full completion of the reaction, L-HPA of high purity (>99% ee) was easily recovered by simple pH shift of the reaction media. This method can permit very efficient asymmetric synthesis of other unnatural amino acids using a single transaminase reaction.     

Asymmetric synthesis of unnaturall-amino acids using thermophilic aromaticl-amino acid transaminase

Aromaticl-amino acid transaminase is an enzyme that is able to transfer the amino group froml-glutamate to unnatural aromatic α-keto acids to generate α-ketoglutarate and unnatural aromaticl-amino acids, respectively. Enrichment culture was used to isolate thermophilicBacillus sp. T30 expressing this enzyme for use in the synthesis of unnaturall-amino acids. The asymmetric syntheses ofl-homophenylalanine andl-phenylglycine resulted in conversion yields of >95% and >93% from 150 mM 2-oxo-4-phenylbutyrate and phenylglyoxylate, respectively, usingl-glutamate as an amino donor at 60°C. Synthesizedl-homophenylalanine andl-phenylglycine were optically pure (>99% enantiomeric excess) and continuously pre-cipitated in the reaction solution due to their low solubility at the given reaction pH. While the solubility of the α-keto acid substrates is dependent on temperature, the solubility of the unnaturall-amino acid products is dependent on the reaction pH. As the solubility difference between substrate and product at the given reaction pH is therefore larger at higher temperature, the thermophilic transaminase was successfully used to shift the reaction equilibrium toward rapid product formation.     

De novo genetic codes and pure translation display

It is appealing to envision engineering translation for the genetically encoded synthesis of new classes of molecules. The complete reassignment of codons to unnatural amino acids at one or two non-adjacent sites per protein has already found wide utility (see other papers in this volume). This has been achieved by suppression at stop codons or rarely used sense codons in crude systems and in vivo. However, competing aminoacyl-tRNAs, aminoacyl-tRNA synthetases, and release factors limit efficiencies and generalization. We maximize flexibility by omitting the competing components and by reconstituting translation from His-tagged initiation and elongation factors. This approach opens up all 64 codons to amino acid reassignment and has allowed incorporation of several adjacent unnatural amino acids for the study of translation mechanism. One potential application is "peptidomimetic evolution" for ligand discovery. Toward this goal, we have demonstrated the display of polypeptides on their mRNAs in a purified translation system, termed "pure translation display."     

Synthetase polyspecificity as a tool to modulate protein function

The site-specific incorporation of unnatural amino acids (UAAs) into proteins in bacteria is made possible by the evolution of aminoacyl-tRNA synthetases that selectively recognize and aminoacylate the amino acid of interest. Recently we have discovered that some of the previously evolved aaRSs display a degree of polyspecificity and are capable of recognizing multiple UAAs. Herein we report the polyspecificity of an aaRS evolved to encode a comarin containing amino acid. This polyspecificity was then exploited to introduce several UAAs into the fluorophore of GFP, altering its photophysical properties.     

An evolved aminoacyl-tRNA synthetase with atypical polysubstrate specificity

We have employed a rapid fluorescence-based screen to assess the polyspecificity of several aminoacyl-tRNA synthetases (aaRSs) against an array of unnatural amino acids. We discovered that a p-cyanophenylalanine specific aminoacyl-tRNA synthetase (pCNF-RS) has high substrate permissivity for unnatural amino acids, while maintaining its ability to discriminate against the 20 canonical amino acids. This orthogonal pCNF-RS, together with its cognate amber nonsense suppressor tRNA, is able to selectively incorporate 18 unnatural amino acids into proteins, including trifluoroketone-, alkynyl-, and halogen-substituted amino acids. In an attempt to improve our understanding of this polyspecificity, the X-ray crystal structure of the aaRS-p-cyanophenylalanine complex was determined. A comparison of this structure with those of other mutant aaRSs showed that both binding site size and other more subtle features control substrate polyspecificity.     

Translational incorporation of multiple unnatural amino acids in a cell-free protein synthesis system

Nature uses 20 canonical amino acids as the standard building blocks of proteins; however, the incorporation of unnatural amino acids (Uaas) can endow polypeptide sequences with new structural and functional features. Although aminoacyl-tRNA synthetases (aaRSs) can accept an array of Uaas in place of their natural counterparts, Uaas generally are charged to tRNAs with substantially lower efficiencies. This particularly makes it difficult to incorporate multiple Uaas into a protein sequence. In this study, we discuss the use of a cell-free protein synthesis system as a versatile platform for the efficient incorporation of multiple Uaas into proteins. Taking advantage of the open nature of cell-free protein synthesis that allows flexible manipulation of its ingredients, we explored the application of Uaas in 10 mM range of concentrations to kinetically overcome the low affinity of aaRSs towards unnatural amino acids. Supplementation of recombinant aaRSs was also investigated to further increase the Uaa-tRNA pools. As a result, under the modified reaction conditions, as many as five different Uaas could be incorporated into a single protein without compromising the yield of protein synthesis.     

S1 pocket fingerprints of human and bacterial methionine aminopeptidases determined using fluorogenic libraries of substrates and phosphorus based inhibitors

Methionyl aminopeptidases (MetAPs) are metallo-dependent proteases responsible for removing of N-terminal methionine residue of peptides and proteins during protein maturation and activation. In this report we use a comprehensive strategy to screen the substrate specificity of three methionyl aminopeptidases: Homo sapiens MetAP-1, Homo sapiens MetAP-2 and Escherichia coli MetAP-1. By utilizing a 65-membered fluorogenic substrate library consisting of natural and unnatural amino acids we established detailed substrate preferences of each enzyme in the S1 pocket. Our results show that this pocket is highly conserved for all investigated MetAPs, very stringent for methionine, and that several unnatural amino acids with methionine-like characteristics were also well hydrolyzed by MetAPs. The substrate-derived results were verified using several phosphonate and phosphinate-based inhibitors.     

Utilization of alkyne bioconjugations to modulate protein function

The ability to introduce or modify protein function has widespread application to multiple scientific disciplines. The introduction of unique unnatural amino acids represents an excellent mechanism to incorporate new functionality; however, this approach is limited by ability of the translational machinery to recognize and incorporate the chemical moiety. To overcome this potential limitation, we aimed to exploit the functionality of existing unnatural amino acids to perform bioorthogonal reactions to introduce the desired protein modification, altering its function. Specifically, via the introduction of a terminal alkyne containing unnatural amino acid, we demonstrated chemically programmable protein modification through the Glaser-Hay coupling to other terminal alkynes, altering the function of a protein. In a proof-of-concept experiment, this approach has been utilized to modify the fluorescence spectrum of green fluorescent protein.     

Cell-free synthesis of angler fish preproinsulin: Complete amino acid sequence of the signal peptide

Messenger ribonucleic acid isolated from angler fish ($\text{Lophius}\text{americanus}$) islets of Langerhans was translated in the wheat germ cell-free protein synthesizing system containing different combinations of radioactive amino acids. Preproinsulin (～ 11,000 daltons) was identified amongst the translation products, by sodium dodecyl sulfate gel electrophoresis, and subjected to microsequencing techniques. The fish preproinsulin was found to possess an NH₂-terminal signal peptide of 24 amino acids, with regions of homology to human, rat and chicken preproinsulin signal sequences.     

Pure translation display

Methods such as monoclonal antibody technology, phage display, and ribosome display provide genetic routes to the selection of proteins and peptides with desired properties. However, extension to polymers of unnatural amino acids is problematic because the translation step is always performed in vivo or in crude extracts in the face of competition from natural amino acids. Here, we address this restriction using a pure translation system in which aminoacyl-tRNA synthetases and other competitors are deliberately omitted. First, we show that such a simplified system can synthesize long polypeptides. Second, we demonstrate "pure translation display" by selecting from an mRNA library only those mRNAs that encode a selectable unnatural amino acid upstream of a peptide spacer sequence long enough to span the ribosome tunnel. Pure translation display should enable the directed evolution of peptide analogs with desirable catalytic or pharmacological properties.     

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.     

Cell-free translation systems for protein engineering

Cell-free translation systems have developed significantly over the last two decades and improvements in yield have resulted in their use for protein production in the laboratory. These systems have protein engineering applications, such as the production of proteins containing unnatural amino acids and development of proteins exhibiting novel functions. Recently, it has been suggested that cell-free translation systems might be used as the fundamental basis for cell-like systems. We review recent progress in the field of cell-free translation systems and describe their use as tools for protein production and engineering.     

Unnatural amino acids as probes of protein structure and function

Nonsense suppression methodology, for incorporating unnatural amino acids into proteins, has enabled a wide range of studies into protein structure and function using both in vitro and in vivo translation systems. Although methodological challenges remain, scores of unnatural amino acids have been employed that include both subtle and dramatic variants of the natural set. A number of insights that would not have been possible using conventional site-directed mutagenesis have been gained.     

Plant Cytochrome P450 Monooxygenases

Plant systems utilize a diverse array of cytochrome P450 monooxygenases (P450s) in their biosynthetic and detoxification pathways. The classic forms of these enzymes are heme-dependent mixed function oxidases that utilize NADPH or NADH and molecular oxygen to produce functionalized organic products. The nonclassical forms are monooxygenases that either do not utilize flavoproteins for dioxygen activation or fail to incorporate molecular oxygen into their final product. Biosynthetic P450s play paramount roles in the synthesis of lignin intermediates, sterols, terpenes, flavonoids, isoflavonoids, furanocoumarins, and a variety of other secondary plant products. Other catabolic P450s metabolize toxic herbicides and insecticides into nontoxic products or, conversely, activate nontoxic substances into toxic products. Biochemical and molecular characterizations on a number of plant P450s have indicated that the relationships between these heme proteins and their substrates are at least as complex as those that exist in mammalian systems. Examples now exist of plant P450s that metabolize: a narrow range of substrates to yield different products, a single substrate to yield different products, multiple substrates to yield the same product, or a single substrate sequentially to yield discrete intermediates in the biosynthesis of a single product. Extensive divergence of catalytic site as well as noncatalytic site residues accounts for the high degree of primary structure variation in the P450 gene superfamily and the diverse array of substrates synthesized and/or detoxified by these proteins. Classic P450s still retain a highly conserved F-G-R-C-G motif in their catalytic site and conserved amino acids in their oxygen binding pocket; nonclassical P450s diverge at several of these positions. A broad range of cloning and transient expression strategies are suitable for plant P450 studies and these have allowed for the isolation and characterization of a number of P450 cDNAs and genes. Because many of these sequences have been cloned only recently, much remains to be learned about the substrate specificities of P450 reactions in plants and the mechanisms by which their genes are regulated.     

Chemical mutagenesis: selective post-expression interconversion of protein amino acid residues

The ability to alter protein structure by site-directed mutagenesis has revolutionized biochemical research. Controlled mutations at the DNA level, before protein translation, are now routine. These techniques allow specific, high fidelity interconversion largely between 20 natural, proteinogenic amino acids. Nonetheless, there is a need to incorporate other amino acids, both natural and unnatural, that are not accessible using standard site-directed mutagenesis and expression systems. Post-translational chemistry offers access to these side chains. Nearly half a century ago, the idea of a 'chemical mutation' was proposed and the interconversion between amino acid side chains was demonstrated on select proteins. In these isolated examples, a powerful proof-of-concept was demonstrated. Here, we revive the idea of chemical mutagenesis and discuss the prospect of its general application in protein science. In particular, we consider amino acids that are chemical precursors to a functional set of other side chains. Among these, dehydroalanine has much potential. There are multiple methods available for dehydroalanine incorporation into proteins and this residue is an acceptor for a variety of nucleophiles. When used in conjunction with standard genetic techniques, chemical mutagenesis may allow access to natural, modified, and unnatural amino residues on translated, folded proteins.