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.     

Protein and cellular engineering with unnatural amino acids

Proteins are the central functional constituents in all living organisms ranging from viruses, bacteria, yeast, and plants to mammals. All of these biopolymers that are formed by natural biosynthetic pathways are composed of a genetically determined sequence of the 20 so-called natural amino acids. The physical and chemical properties of proteins are a reflection of the side chains of each of the component amino acids. However, for some purposes it would be very desireable to have amino acids with side chains of various selected physical chemical properties, such as a keto group, a crosslinker, or a NMR probe group, incorporated into the protein. Although chemical and biochemical methods for modifying amino acid moieties in proteins have been achieved, recent successes in incorporating unnatural amino acids in vivo open entirely new avenues for determining protein functions in vivo and for the creation of unnatural proteins with novel functionalities. Several examples by employing the novel activity of unnatural amino acids have shown significant roles in both basic research and biotechnology.     

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.     

Enzymatic tRNA acylation by acid and alpha-hydroxy acid analogues of amino acids

Incorporation of unnatural amino acids with unique chemical functionalities has proven to be a valuable tool for expansion of the functional repertoire and properties of proteins as well as for structure-function analysis. Incorporation of alpha-hydroxy acids (primary amino group is substituted with hydroxyl) leads to the synthesis of proteins with peptide bonds being substituted by ester bonds. Practical application of this modification is limited by the necessity to prepare corresponding acylated tRNA by chemical synthesis. We investigated the possibility of enzymatic incorporation of alpha-hydroxy acid and acid analogues (lacking amino group) of amino acids into tRNA using aminoacyl-tRNA synthetases (aaRSs). We studied direct acylation of tRNAs by alpha-hydroxy acid and acid analogues of amino acids and corresponding chemically synthesized analogues of aminoacyl-adenylates. Using adenylate analogues we were able to enzymatically acylate tRNA with amino acid analogues which were otherwise completely inactive in direct aminoacylation reaction, thus bypassing the natural mechanisms ensuring the selectivity of tRNA aminoacylation. Our results are the first demonstration that the use of synthetic aminoacyl-adenylates as substrates in tRNA aminoacylation reaction may provide a way for incorporation of unnatural amino acids into tRNA, and consequently into proteins.     

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.     

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.     

Synthesis,photophysical properties of triazolyl-donor/acceptor chromophores decorated unnatural amino acids: Incorporation of a pair into Leu-enkephalin peptide and application of triazolylperylene amino acid in sensing BSA

The research in the field of design and synthesis of unnatural amino acids is growing at a fast space for the increasing demand of proteins of potential therapeutics and many other diversified novel functional applications. Thus, we report herein the design and synthesis of microenvironment sensitive fluorescent triazolyl unnatural amino acids (UNAA) decorated with donor and/or acceptor aromatic chromophores via click chemistry. The synthesized fluorescent amino acids show interesting solvatochromic characteristic and/or intramolecular charge transfer (ICT) feature as is revealed from the UV–visible, fluorescence photophysical properties and DFT/TDDFT calculation. HOMO–LUMO distribution shows that the emissive states of some of the amino acids are characterized with more significant electron redistribution between the triazolyl moiety and the aromatic chromophores linked to it leading to modulated emission property. A pair of donor–acceptor amino acid shows interesting photophysical interaction property indicating a FRET quenching event. Furthermore, one of the amino acid, triazolyl-perylene amino acid, has been exploited for studying interaction with BSA and found that it is able to sense BSA with an enhancement of fluorescence intensity. Finally, we incorporated a pair of donor/acceptor amino acids into a Leu-enkephalin analogue pentapeptide which was found to adopt predominantly type II β-turn conformation. We envisage that our investigation is of importance for the development of new fluorescent donor–acceptor unnatural amino acids a pair of which can be exploited for generating fluorescent peptidomimetic probe of interesting photophysical property for applications in studying peptide–protein interaction.     

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.     

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.     

Creation,Optimization, and Use of Semi-Synthetic Organisms that Store and Retrieve Increased Genetic Information

With few exceptions, natural proteins are built from only 20 canonical (proteogenic) amino acids which limits the functionality and accordingly the properties they can possess. Genetic code expansion, i.e. the creation of codons and the machinery needed to assign them to non-canonical amino acids (ncAAs), promises to enable the discovery of proteins with novel properties that are otherwise difficult or impossible to obtain. One approach to expanding the genetic code is to expand the genetic alphabet via the development of unnatural nucleotides that pair to form an unnatural base pair (UBP). Semi-synthetic organisms (SSOs), i.e. organisms that stably maintain the UBP, transcribe its component nucleotides into RNA, and use it to translate proteins, would have available to them new codons and the anticodons needed to assign them to ncAAs. This review summarizes the development of a family of UBPs, their use to create SSOs, and the optimization and application of the SSOs to produce candidate therapeutic proteins with improved properties that are now undergoing evaluation in clinical trials.     

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.     

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.     

Expanding the genetic code of Drosophila melanogaster

Genetic code expansion for unnatural amino acid mutagenesis has, until recently, been limited to cell culture. We demonstrate the site-specific incorporation of unnatural amino acids into proteins in Drosophila melanogaster at different developmental stages, in specific tissues and in a subset of cells within a tissue. This approach provides a foundation for probing and controlling processes in this established metazoan model organism with a new level of molecular precision.     

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.     

Unnatural amino acid mutagenesis: a precise tool for probing protein structure and function

The first general method for the biosynthetic incorporation of unnatural amino acids into proteins was reported in 1989. The ensuing years have seen the solid development and subsequent implementation of "unnatural amino acid mutagenesis" in a number of groundbreaking studies. Over 100 different amino acids have been incorporated into dozens of soluble and transmembrane proteins, using both cell-extract and cell-intact translation systems. The approach has provided insights into ligand-binding sites, conformational changes, and protein-protein interactions with a level of precision simply unparalleled by conventional mutagenesis. Here, the methodology is outlined, significant applications of the approach are summarized, and recent major improvements in the method are discussed. The future will likely see many more investigators utilizing this approach to manipulate proteins as it realizes its promise of becoming a tool with enormous potential.     

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.     

Effect of temperature and ATP supply on the efficiency of programmed nonsense suppression

Chemical diversity of protein molecules can be expanded through in vitro incorporation of unnatural amino acids in response to a nonsense codon. Chemically misacylated tRNAs are used for tethering unnatural amino acids to a nonsense-mutated target codon (nonsense suppression). In the course of experiments to introduce S-(2-nitrobenzyl)cysteine (NBC) into a targeted location of human erythropoietin, we found that NBC incorporates more efficiently at lower temperatures. In addition, at a fixed reaction temperature, more NBC was incorporated with a reduced supply of ATP. Since the rate of peptide elongation was remarkably higher at the elevated temperature or with enhanced supply of ATP, these results indicate that the efficiency of nonsense suppression is inversely correlated to the peptide elongation rate. Therefore, maximal yield of nonsense-suppressed proteins is obtained at a compromised elongation rate. The present result will offer a primary guideline to optimize the reaction conditions for in vitro production of protein molecules containing unnatural amino acids.     

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.