The use of 5''-phospho-2 deoxyribocytidylylriboadenosine as a facile route to chemical aminoacylation of tRNA.

Methodology is described for the synthesis and chemical aminoacylation of the hybrid dinucleotide 5'-phospho-2'-deoxyribocytidylylriboadenosine (pdCpA). Ligation of aminoacylated pdCpA to a truncated amber suppressor tRNACUA (-CA) using T4 RNA ligase generates an aminoacylated suppressor tRNA which can be used for site-specific incorporation of unnatural amino acids into proteins. Both the ligation and in vitro suppression efficiencies are the same when either pCpA or pdCpA is used. The use of deoxycytidine simplifies the chemistry involved in the synthesis of the dinucleotide pCpA. In addition, these results demonstrate that ribocytidine is not required for recognition of the aminoacylated tRNA during protein synthesis.     

Phage selection for site-specific incorporation of unnatural amino acids into proteins in vivo.

The development of a method for the site-specific incorporation of unnatural amino acids into proteins in vivo would significantly facilitate studies of the cellular function of proteins, as well as make possible the synthesis of proteins with novel structures and activities. Our approach to this problem consists of the generation of amber suppressor tRNA/aminoacyl-tRNA synthetase pairs that are not catalytically competent with all the endogenous Escherichia coli tRNAs and aminoacyl-tRNA synthetases, followed by directed evolution of such orthogonal aminoacyl-tRNA synthetases to alter their amino acid specificities. To evolve the desired amino acid specificity, a direct selection for site-specific incorporation of unnatural amino acids into a reporter epitope displayed on the surface of M13 phage has been developed and characterized. Under simulated selection conditions, phage particles displaying aspartate were enriched over 300-fold from a pool of phage displaying asparagine using monoclonal antibodies raised against the aspartate-containing epitope. The direct phage selection offers high specificity for the amino acid of interest, eliminating the potential for contamination with synthetases active towards wild-type amino acids in multiple rounds of selection.     

Transferability of N-terminal mutations of pyrrolysyl-tRNA synthetase in one species to that in another species on unnatural amino acid incorporation efficiency

Genetic code expansion is a powerful technique for site-specific incorporation of an unnatural amino acid into a protein of interest. This technique relies on an orthogonal aminoacyl-tRNA synthetase/tRNA pair and has enabled incorporation of over 100 different unnatural amino acids into ribosomally synthesized proteins in cells. Pyrrolysyl-tRNA synthetase (PylRS) and its cognate tRNA from Methanosarcina species are arguably the most widely used orthogonal pair. Here, we investigated whether beneficial effect in unnatural amino acid incorporation caused by N-terminal mutations in PylRS of one species is transferable to PylRS of another species. It was shown that conserved mutations on the N-terminal domain of MmPylRS improved the unnatural amino acid incorporation efficiency up to five folds. As MbPylRS shares high sequence identity to MmPylRS, and the two homologs are often used interchangeably, we examined incorporation of five unnatural amino acids by four MbPylRS variants at two temperatures. Our results indicate that the beneficial N-terminal mutations in MmPylRS did not improve unnatural amino acid incorporation efficiency by MbPylRS. Knowledge from this work contributes to our understanding of PylRS homologs which are needed to improve the technique of genetic code expansion in the future.

     

Fidelity in the aminoacylation of tRNA(Val) with hydroxy analogues of valine, leucine, and isoleucine by valyl-tRNA synthetases from Saccharomyces cerevisiae and Escherichia coli

Several analogues of valine, leucine, and isoleucine carrying hydroxyl groups in the gamma- or delta-position have been tested in the aminoacylation of tRNA by valyl-tRNA synthetases from Saccharomyces cerevisiae and Escherichia coli. Results of the ATP/PPi exchange and of the aminoacylation reactions indicate that the amino acid analogues not only can form the aminoacyl adenylate intermediate but are also transferred to tRNA. However, the fact that the reaction consumes an excess of ATP indicates that the misactivated amino acid analogue is hydrolytically removed. Thus, valyl-tRNA synthetase from S. cerevisiae shows a high fidelity in forming valyl-tRNA. Although the much bulkier amino acid analogues allo- and iso-gamma-hydroxyvaline and allo- and iso-gamma-hydroxyisoleucine are initially charged to tRNA, the misaminoacylated tRNA(Val) is enzymatically deacylated. This cleavage reaction is mediated by the hydroxyl groups of the amino acid analogues which are converted into the corresponding lactones.     

Biochemical research on oogenesis: protein synthesis by purified 42S particles from Xenopus laevis and Tinca tinca previtellogenic oocytes

When incubated with ATP and a labeled amino acid, the 42S particles from early oocytes of Xenopus laevis and Tinca tinca incorporate radioactivity into tRNA and into a high molecular mass material which can be identified as protein. This incorporation is totally independent of ribosomes of cytosolic, mitochondrial or bacterial origin. The incorporated amino acids are linked to a broad spectrum of proteins by covalent bonds. Simple treatments such as incubation in buffer or addition of synthetic polyribonucleotides can inhibit the protein-labeling activity of the particles without affecting their tRNA aminoacylation activity. The former activity corresponds either to an amino acid polymerization reaction or to a protein-modifying reaction of a novel type. No involvement of mRNA in this process has been demonstrated. The alleged amino acid polymerization activity of the 42S particles could be a consequence of the conditions provided to aminoacyl tRNA by the tRNA-binding sites of the particles. These conditions are likely to allow the peptidyl transfer reaction to take place, although at a much lower rate than in the ribosome.     

Effects of p-chlorophenylalanine and alpha-methylphenylalanine on amino acid uptake and protein synthesis in mouse neuroblastoma cells.

The phenylalanine analogues p-chlorophenylalanine and alpha-methylphenylalanine were used to inhibit phenylalanine hydroxylase in animal models for phenylketonuria. The present report examines the affects of these analogues on the metabolism of neuroblastoma cells. p-Chlorophenylalanine inhibited growth and was toxic to neuroblastoma cells. Although in vivo this analogue increased cell monoribosomes by 42%, it did not significantly affect poly(U)-directed protein synthesis in vitro. P-Chlorophenylalanine did not compete with phenylalanine or tyrosine for aminoacylation of tRNA and was therefore not substituted for those amino acids in nascent polypeptides. The initial cellular uptake of various large neutral amino acids was inhibited by this analogue but did not affect the flux of amino acids already in the cell; this suggested that an alteration of the cell's amino acid pools was not responsible for the cytotoxicity of the analogues. In contrast with p-chlorophenylalanine, alpha-methylphenylalanine did not exert these direct toxic effects because the administration of alpha-methylphenylalanine in vivo did not affect brain polyribosomes and a comparable concentration of this analogue was neither growth inhibitory nor cytotoxic to neuroblastoma cells in culture. The suitability of each analogue as an inhibitor of phenylalanine hydroxylase in animal models for phenylketonuria is discussed.     

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.     

An efficient protocol for incorporation of an unnatural amino acid in perdeuterated recombinant proteins using glucose-based media

The in vivo incorporation of unnatural amino acids into proteins is a well-established technique requiring an orthogonal tRNA/aminoacyl-tRNA synthetase pair specific for the unnatural amino acid that is incorporated at a position encoded by a TAG amber codon. Although this technology provides unique opportunities to engineer protein structures, poor protein yields are usually obtained in deuterated media, hampering its application in the protein NMR field. Here, we describe a novel protocol for incorporating unnatural amino acids into fully deuterated proteins using glucose-based media (which are relevant to the production, for example, of amino acid-specific methyl-labeled proteins used in the study of large molecular weight systems). The method consists of pre-induction of the pEVOL plasmid encoding the tRNA/aminoacyl-tRNA synthetase pair in a rich, H₂O-based medium prior to exchanging the culture into a D₂O-based medium. Our protocol results in high level of isotopic incorporation (~95%) and retains the high expression level of the target protein observed in Luria–Bertani medium.     

Synthesis of alanyl nucleobase amino acids and their incorporation into proteins

Proteins which bind to nucleic acids and regulate their structure and functions are numerous and exceptionally important. Such proteins employ a variety of strategies for recognition of the relevant structural elements in their nucleic acid substrates, some of which have been shown to involve rather subtle interactions which might have been difficult to design from first principles. In the present study, we have explored the preparation of proteins containing unnatural amino acids having nucleobase side chains. In principle, the introduction of multiple nucleobase amino acids into the nucleic acid binding domain of a protein should enable these modified proteins to interact with their nucleic acid substrates using Watson-Crick and other base pairing interactions. We describe the synthesis of five alanyl nucleobase amino acids protected in a fashion which enabled their attachment to a suppressor tRNA, and their incorporation into each of two proteins with acceptable efficiencies. The nucleobases studied included cytosine, uracil, thymine, adenine and guanine, i.e. the major nucleobase constituents of DNA and RNA. Dihydrofolate reductase was chosen as one model protein to enable direct comparison of the facility of incorporation of the nucleobase amino acids with numerous other unnatural amino acids studied previously. The Klenow fragment of DNA polymerase I was chosen as a representative DNA binding protein whose mode of action has been studied in detail.     

Site-specific incorporation of (aminooxy)acetic acid into proteins

By employing a general biosynthetic method for the elaboration of proteins containing unnatural amino acid analogues, we incorporated (aminooxy)acetic acid into positions 10 and 27 of Escherichia coli dihydrofolate reductase. Introduction of the modified amino acid into DHFR was accomplished in an in vitro protein biosynthesizing system by readthrough of a nonsense (UAG) codon with a suppressor tRNA that had been activated with (aminooxy)acetic acid. Incorporation of the amino acid proceeded with reasonable efficiency at codon position 10 but less well at position 27. (Aminooxy)acetic acid was also incorporated into position 72 of DNA polymerase beta. Peptides containing (aminooxy)acetic acid have been shown to adopt a preferred conformation involving an eight-membered ring that resembles a gamma-turn. Accordingly, the present study may facilitate the elaboration of proteins containing conformationally biased peptidomimetic motifs at predetermined sites. The present results further extend the examples of ribosomally mediated formation of peptide bond analogues of altered connectivity and provide a conformationally biased linkage at a predetermined site. It has also been shown that the elaborated protein can be cleaved chemically at the site containing the modified amino acid.     

Improved amber and opal suppressor tRNAs for incorporation of unnatural amino acids in vivo. Part 1: minimizing misacylation

The incorporation of unnatural amino acids site-specifically is a valuable technique for structure-function studies, incorporation of biophysical probes, and determining protein-protein interactions. THG73 is an amber suppressor tRNA used extensively for the incorporation of >100 different residues in over 20 proteins, but under certain conditions THG73 is aminoacylated in vivo by endogenous aminoacyl-tRNA synthetase. Similar aminoacylation is seen with the Escherichia coli Asn amber suppressor tRNA, which has also been used to incorporate UAAs in many studies. We now find that the natural amino acid placed on THG73 is Gln. Since the E. coli GlnRS recognizes positions in the acceptor stem, we made several acceptor stem mutations in the second to fourth positions on THG73. All mutations reduce aminoacylation in vivo and allow for the selection of highly orthogonal tRNAs. To show the generality of these mutations, we created opal suppressor tRNAs that show less aminoacylation in Xenopus oocytes relative to THG73. We have created a library of Tetrahymena thermophila Gln amber suppressor tRNAs that will be useful for determining optimal suppressor tRNAs for use in other eukaryotic cells.     

Misacylation of yeast amber suppressor tRNA(Tyr) by E. coli lysyl-tRNA synthetase and its effective repression by genetic engineering of the tRNA sequence

Through an exhaustive search for Escherichia coli aminoacyl-tRNA synthetase(s) responsible for the misacylation of yeast suppressor tRNA(Tyr), E. coli lysyl-tRNA synthetase was found to have a weak activity to aminoacylate yeast amber suppressor tRNA(Tyr) (CUA) with L-lysine. Since our protein-synthesizing system for site-specific incorporation of unnatural amino acids into proteins is based on the use of yeast suppressor tRNA(Tyr)/tyrosyl-tRNA synthetase (TyrRS) pair as the "carrier" of unusual amino acid in E. coli translation system, this misacylation must be repressed as low as possible. We have succeeded in effectively repressing the misacylation by changing several nucleotides in this tRNA by genetic engineering. This "optimized" tRNA together with our mutant TyrRS should serve as an efficient and faithful tool for site-specific incorporation of unnatural amino acids into proteins in a protein-synthesizing system in vitro or in vivo.     

Four-base codon/anticodon strategy and non-enzymatic aminoacylation for protein engineering with non-natural amino acids

Techniques for position-specific incorporation of non-natural amino acids in an in vitro protein synthesizing system are described. First, a PNA-assisted non-enzymatic tRNA aminoacylation with a variety of natural and non-natural amino acids is described. With this technique, one can aminoacylate a specific tRNA simply by adding a preformed amino acid activated ester-PNA conjugate into an in vitro protein biosynthesizing system. Second, the genetic code is expanded by introducing 4-base codons that can be exclusively translated to non-natural amino acids. The most advantageous point of the 4-base codon strategy is to introduce multiple amino acids into specific positions in single proteins by using mutually orthogonal 4-base codons and orthogonal tRNAs. An easy and quick method for preparation of tRNAs possessing 4-base anticodons is also described. Combination of the non-enzymatic aminoacylation and the 4-base codon/anticodon strategy gives an easy and widely applicable technique for incorporating a variety of non-natural amino acids into proteins in vitro.     

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.     

Genetic introduction of a diketone-containing amino acid into proteins

An orthogonal tRNA/aminoacyl-tRNA synthetase pair was evolved that makes possible the site-specific incorporation of an unnatural amino acid bearing a beta-diketone side chain into proteins in Escherichia coli with high translational efficiency and fidelity. Proteins containing this unnatural amino acid can be efficiently and selectively modified with hydroxylamine derivatives of fluorophores and other biophysical probes.     

Misincorporation of amino acid analogues into proteins by biosynthesis

Despite astounding diversity in their structure and function, proteins are constructed from 22 protein or ‘canonical’ amino acids. Hundreds of amino acid analogues exist; many occur naturally in plants, some are synthetically produced or can be produced in vivo by oxidation of amino acid side-chains. Certain structural analogues of the protein amino acids can escape detection by the cellular machinery for protein synthesis and become misincorporated into the growing polypeptide chain of proteins to generate non-native proteins. In this review we seek to provide a comprehensive overview of the current knowledge on the biosynthetic incorporation of amino acid analogues into proteins by mammalian cells. We highlight factors influencing their incorporation and how the non-native proteins generated can alter cell function. We examine the ability of amino acid analogues, representing those commonly found in damaged proteins in pathological tissues, to be misincorporated into proteins by cells in vitro, providing us with a useful tool in the laboratory to generate modified proteins representing those present in a wide-range of pathologies. We also discuss the evidence for amino acid analogue incorporation in vivo and its association with autoimmune symptoms. We confine the review to studies in which the synthetic machinery of cell has not been modified to accept non-protein amino acids.     

Aminoacylation of undermethylated mammalian transfer RNA.

To study the role of 5-methylcytidine in the aminoacylation of mammalian tRNA, bulk tRNA specifically deficient in 5-methylcytidine was isolated from the livers of mice treated with 5-azacytidine (18 mg/kg) for 4 days. For comparison, more extensively altered tRNA was isolated from the livers of mice treated with DL-ethionine (100 mg/kg) plus adenine (48 mg/kg) for 3 days. The amino acid acceptor capacity of these tRNAs was determined by measuring the incorporation of one of eight different 14C-labeled amino acids or a mixture of 14C-labeled amino acids in homologous assays using a crude synthetase preparation isolated from untreated mice. The 5-methylcytidine-deficient tRNA incorporated each amino acid to the same extent as fully methylated tRNA. The tRNA from DL-ethionine-treated livers showed an overall decreased amino-acylation capacity for all amino acids tested. The 5-methylcytidine-deficient tRNA from DL-ethionine-treated mice were further characterized as substrates in homologous rate assays designed to determine the Km and V of the aminoacylation reaction using four individual 14C-labeled amino acids and a mixture of 14C-labeled amino acids. The Km and V of the reactions for all amino acids tested using 5-methylcytidine-deficient tRNA as substrate were essentially the same as for fully methylated tRNA. However, the Km and V were increased when liver tRNA from mice treated with DL-ethionine plus adenine was used as substrate in the rate reaction with [14C]lysine as label. Our results suggest that although extensively altered tRNA is a poorer substrate than control tRNA in both extent and rate of aminoacylation, 5-methylcytidine in mammalian tRNA is not involved in the recognition of the tRNA by the synthetase as measured by aminoacylation activity.     

Importance of expression system in the production of unnatural recombinant proteins in Escherichia coli

In this study, we investigated the efficiencies by which the pET and pQE expression systems produce unnatural recombinant proteins by residue-specific incorporation of unnatural amino acids, a method through which it was found that type of gene expression system tremendously influences the production yield of unnatural proteins in Escherichia coli. Green fluorescent protein (GFP) and a single-chain Fv antibody against c-Met were utilized as model recombinant proteins while L-homopropargylglycine (Hpg), a methionine analogue that incorporates into the methionine residues of a recombinant protein, was used as model unnatural amino acid. The pET system produced an almost negligible amount of Hpg-incorporated unnatural protein compared to the amount of methionine-incorporated natural protein. However, comparable amounts of unnatural and natural protein were produced by the pQE expression system. The amount of unnatural GFP protein produced through pET expression was not increased despite the over-expression of methionyl tRNA synthetase, which can enhance the activation rate of methionyl-tRNA with a methionine analogue. Incorporation of Hpg decreased the productivity of active GFP by approximately 2.5 fold, possibly caused by the inefficient folding of Hpg-incorporated GFP. Conversely, the productivity of functional anti-c-Met sc-Fv was not influenced by incorporation of Hpg. We confirmed through LC-MS and LCMS/MS that Hpg was incorporated into the methionine residues of the recombinant proteins produced by the pQE expression system. The first two authors equally contributed to this work.     

An archaebacteria-derived glutamyl-tRNA synthetase and tRNA pair for unnatural amino acid mutagenesis of proteins in Escherichia coli

The addition of novel amino acids to the genetic code of Escherichia coli involves the generation of an aminoacyl-tRNA synthetase and tRNA pair that is ‘orthogonal’, meaning that it functions independently of the synthetases and tRNAs endogenous to E.coli. The amino acid specificity of the orthogonal synthetase is then modified to charge the corresponding orthogonal tRNA with an unnatural amino acid that is subsequently incorporated into a polypeptide in response to a nonsense or missense codon. Here we report the development of an orthogonal glutamic acid synthetase and tRNA pair. The tRNA is derived from the consensus sequence obtained from a multiple sequence alignment of archaeal tRNA^Glu sequences. The glutamyl-tRNA synthetase is from the achaebacterium Pyrococcus horikoshii. The new orthogonal pair suppresses amber nonsense codons with an efficiency roughly comparable to that of the orthogonal tyrosine pair derived from Methanococcus jannaschii, which has been used to selectively incorporate a variety of unnatural amino acids into proteins in E.coli. Development of the glutamic acid orthogonal pair increases the potential diversity of unnatural amino acid structures that may be incorporated into proteins in E.coli.