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.     

Effects of release factor 1 on in vitro protein translation and the elaboration of proteins containing unnatural amino acids.

An in vitro protein synthesizing system was modified to facilitate the improved, site-specific incorporation of unnatural amino acids into proteins via readthrough of mRNA nonsense (UAG) codons by chemically misacylated suppressor tRNAs. The modified system included an S-30 extract derived from Escherichia coli that expresses a temperature-sensitive variant of E. coli release factor 1 (RF1). Mild heat treatment of the S-30 extract partially deactivated RF1 and improved UAG codon readthrough by as much as 11-fold, as demonstrated by the incorporation of unnatural amino acids into positions 25 and 125 of HIV-1 protease and positions 10 and 22 of E. coli dihydrofolate reductase. The increases in yields were the greatest for those amino acids normally incorporated poorly in the in vitro protein synthesizing system, thus significantly enhancing the repertoire of modified amino acids that can be incorporated into the proteins of interest. The substantial increase in mutant protein yields over those obtained with an S-30 extract derived from an RF1 proficient E. coli strain is proposed to result from a relaxed stringency of termination by RF1 at the stop codon (UAG). When RF1 levels were depleted further, the intrinsic rate of DHFR synthesis increased, consistent with the possibility that RF1 competes not only at stop codons but also at other mRNA codons during peptide elongation. It thus seems possible that in addition to its currently accepted role as a protein factor involved in peptide termination, RF1 is also involved in functions that control the rate at which protein synthesis proceeds.     

Unnatural RNA display libraries

Combinatorial peptide and protein libraries have now been developed to accommodate unnatural amino acids in a genetically encoded format via in vitro nonsense and sense suppression. General translation features and specific regioselective and stereoselective properties of the ribosome endow these libraries with a broad chemical diversity. Alternatively, amino acid residues can be chemically derivatized post-translationally to add preferred functionality to the encoded peptide. All of these efforts are advancing combinatorial peptide and protein libraries for enhanced ligands against biological targets of interest.     

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。     

A wheat embryo cell‐free protein synthesis system not requiring an exogenous supply of GTP

Most in vitro protein synthesis systems require a supply of GTP for the formation of translation initiation complexes, with two GTP molecules per amino acid needed as an energy source for a peptide elongation reaction. In order to optimize protein synthesis reactions in a continuous‐flow wheat embryo cell‐free system, we have examined the influence of adding GTP and found that the system does not require any supply of GTP. We report here the preparation of a wheat embryo extract from which endogenous GTP was removed by gel filtration, and the influence of adding GTP to the system on protein synthesis reactions. Using Green Fluorescent Protein (GFP) as a reporter, higher levels of production were observed at lower concentrations of GTP, with the optimal level of production obtained with no supply of GTP. A HPLC‐based analysis of the extract and the translation mixture containing only ATP as an energy source revealed that GTP was not detectable in the extract, however, 35 μM of GTP was found in the translation mixture. This result suggests that GTP could be generated from other compounds, such as GDP and GMP, using ATP. A similar experiment with a C‐terminally truncated form of human protein tyrosine phosphatase 1B (hPTP1B_1‐320) gave almost the same result. The wheat embryo cell‐free translation system worked most efficiently without exogenous GTP, producing 3.5 mg/mL of translation mixture over a 48‐h period at 26°C. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

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.     

Chemically mediated site-specific proteolysis. Alteration of protein-protein interaction

The design and synthesis of a novel iodine-labile serine protease inhibitor was realized by the use of an ecotin analogue containing allylglycine at position 84 in lieu of methionine. Allylglycine-containing ecotins were synthesized by in vitro translation of the ecotin gene containing an engineered nonsense codon (TAG) at the positions of interest. A misacylated suppressor tRNA activated with the unnatural amino acid allylglycine was employed for the suppression of the nonsense codons in a cell-free protein biosynthesizing system, permitting the elaboration of ecotin analogues containing allyglycine at the desired sites. The derived ecotin analogues were capable of inhibiting bovine trypsin with inhibitory constants (K(i)s) comparable to that of wild-type ecotin. Iodine treatment of ecotin analogue Met84(A)Gly resulted in the deactivation of ecotin, caused by peptide backbone cleavage at its P1 reactive site. Upon iodine treatment, active trypsin could be released from the protein complex with ecotin analogue Met84(A)Gly. This constitutes a novel strategy for modulation of serine protease activity and more generally for alteration of protein-protein interaction by a simple chemical reagent.     

Enhanced Yield of Recombinant Proteins with Site-Specifically Incorporated Unnatural Amino Acids Using a Cell-Free Expression System

Using a commercial protein expression system, we sought the crucial elements and conditions for the expression of proteins with genetically encoded unnatural amino acids. By identifying the most important translational components, we were able to increase suppression efficiency to 55% and to increase mutant protein yields to levels higher than achieved with wild type expression (120%), reaching over 500 µg/mL of translated protein (comprising 25 µg in 50 µL of reaction mixture). To our knowledge, these results are the highest obtained for both in vivo and in vitro systems. We also demonstrated that efficiency of nonsense suppression depends greatly on the nucleotide following the stop codon. Insights gained in this thorough analysis could prove useful for augmenting in vivo expression levels as well.     

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.     

Adaptation of an orthogonal archaeal leucyl-tRNA and synthetase pair for four-base,amber, and opal suppression

Recently, it has been shown that an amber suppressor tRNA/aminoacyl-tRNA synthetase pair derived from the tyrosyl-tRNA synthetase of Methanococcus jannaschii can be used to genetically encode unnatural amino acids in response to the amber nonsense codon, TAG. However, we have been unable to modify this pair to decode either the opal nonsense codon, TGA, or the four-base codon, AGGA, limiting us to a 21 amino acid code. To overcome this limitation, we have adapted a leucyl-tRNA synthetase from Methanobacterium thermoautotrophicum and leucyl tRNA derived from Halobacterium sp. NRC-1 as an orthogonal tRNA-synthetase pair in Escherichia coli to decode amber (TAG), opal (TGA), and four-base (AGGA) codons. To improve the efficiency and selectivity of the suppressor tRNA, extensive mutagenesis was performed on the anticodon loop and acceptor stem. The two most significant criteria required for an efficient amber orthogonal suppressor tRNA are a CU(X)XXXAA anticodon loop and the lack of noncanonical or mismatched base pairs in the stem regions. These changes afford only weak suppression of TGA and AGGA. However, this information together with an analysis of sequence similarity of multiple native archaeal tRNA sequences led to efficient, orthogonal suppressors of opal codons and the four-base codon, AGGA. Ultimately, it should be possible to use these additional orthogonal pairs to genetically incorporate multiple unnatural amino acids into proteins.     

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 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.     

Studies of the chemical basis of the origin of protein synthesis: Initiation and direction of peptide growth

Summary The data presented in this paper show that the ease of non-enzymatic activation of carboxylic acids by ATP at pH 5 varies directly with the pK_a of the carboxyl group, and is consistent with the idea that it is the protonated form of the carboxyl group which participates in the activation reaction. Consequently, since most N-blocked amino acids have higher pK_a's than do their unblocked forms, they are activated more readily, and we have demonstrated that this principle applies to peptides as well,which are activated more rapidly than single amino acids. We propose that this fact may be partly responsible for the origin of two important features still observed in contemporary protein synthesis: (1) initiation in prokaryotes is accomplished with an N-blocked amino acid, and (2) elongation in all living systems occurs at the carboxyl end of the growing peptide.     

Cloning and characterization of the nuclear AC115 gene of Chlamydomonas reinhardtii

The nuclear ac115 mutant of Chlamydomonas reinhardtii is specifically blocked in the synthesis of the chloroplast encoded D2 protein of the photosystem II reaction center at a point after translation initiation. Here, we report the identification of the AC115 gene through complementation rescue of the ac115 mutant strain, using an indexed cosmid library of Chlamydomonas genomic DNA. AC115 is a small, novel, intronless nuclear gene which encodes a protein of 113 amino acids. The amino terminal end of the Ac115 protein is rich in basic amino acids and has features which resemble a chloroplast transit sequence. A hydrophobic stretch of amino acids at the protein's carboxyl terminus is sufficiently large to be a membrane spanning or a protein/protein interaction domain. Various models are discussed to account for the mechanism by which Ac115p works in D2 synthesis. The ac115 mutant allele was sequenced and determined to be an A-to-T transversion at the first position of the fourth codon of the coding sequence. This mutation changes an AAG codon to a TAG nonsense codon and results in a null phenotype.     

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."     

Synthesis of novel unnatural amino acid as a building block and its incorporation into an antimicrobial peptide

Considering the biological mechanism and in vivo stability of antimicrobial peptides, we designed and synthesized novel unnatural amino acids with more positively charged and bulky side chain group than lysine residue. The unusual amino acids, which were synthesized by either solution phase or solid phase, were incorporated into an antimicrobial peptide. Its effect on the stability, activity, and the structure of the peptide was studied to evaluate the potential of these novel unnatural amino acids as a building block for antimicrobial peptides. The incorporation of this unusual amino acid increased the resistance of the peptide against serum protease more than three times without a decrease in the activity. Circular dichroism spectra of the peptides indicated that all novel unnatural amino acids must have lower helical forming propensities than lysine. Our results indicated that the unnatural amino acids synthesized in this study could be used not only as a novel building block for combinatorial libraries of antimicrobial peptides, but also for structure–activity relationship studies about antimicrobial peptides.     

mRNA display: ligand discovery,interaction analysis and beyond

In vitro peptide and protein selection using mRNA display enables the discovery and directed evolution of new molecules from combinatorial libraries. These selected molecules can serve as tools to control and understand biological processes, enhance our understanding of molecular interactions and potentially treat disease in therapeutic applications. In mRNA display, mRNA molecules are covalently attached to the peptide or protein they encode. These mRNA-protein fusions enable in vitro selection of peptide and protein libraries of >10(13) different sequences. mRNA display has been used to discover novel peptide and protein ligands for RNA, small molecules and proteins, as well as to define cellular interaction partners of proteins and drugs. In addition, several unique applications are possible with mRNA display, including self-assembling protein chips and library construction with unnatural amino acids and chemically modified peptides.     

Dynamic Model of the Process of Protein Synthesis in Eukaryotic Cells

Protein synthesis is the final step of gene expression in all cells. In order to understand the regulation of this process, it is important to have an accurate model that incorporates the regulatory steps. The model presented in this paper is composed of set of differential equations which describe the dynamics of the initiation process and its control, as well as peptide elongation, starting with the amino acids available for peptide creation. A novel approach for modeling the elongation process permits useful prediction of protein production and consumption of energy and amino acids, as well as ribosome loading rate and ribosome spacing on the mRNA. An erratum to this article can be found at     

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.     

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.