Eukaryotic Cell-free Translation System Research Articles

Cell Engineering and Cultivation of Chinese Hamster Ovary Cells for the Development of Orthogonal Eukaryotic Cell-free Translation Systems.

The investigation of protein structures, functions and interactions often requires modifications to adapt protein properties to the specific application. Among many possible methods to equip proteins with new chemical groups, the utilization of orthogonal aminoacyl-tRNA synthetase/tRNA pairs enables the site-specific incorporation of non-canonical amino acids at defined positions in the protein. The open nature of cell-free protein synthesis reactions provides an optimal environment, as the orthogonal components do not need to be transported across the cell membrane and the impact on cell viability is negligible. In the present work, it was shown that the expression of orthogonal aminoacyl-tRNA synthetases in CHO cells prior to cell disruption enhanced the modification of the pharmaceutically relevant adenosine A2a receptor. For this purpose, in complement to transient transfection of CHO cells, an approach based on CRISPR/Cas9 technology was selected to generate a translationally active cell lysate harboring endogenous orthogonal aminoacyl-tRNA synthetase.

Suppressor tRNA-based Biosensors for Detecting Analytes.

A nonsense suppressor tRNA (sup-tRNA) allows a natural or non-natural amino acid to be assigned to a nonsense codon in mRNA. Sup-tRNAs were utilized initially for studying tRNA functions but lately are used more for protein engineering and gene regulation. In the latter application, a sup-tRNA that is aminoacylated with a natural amino acid by the corresponding aminoacyl-tRNA synthetase is used to express a full-length natural protein from its mutated gene with a nonsense codon in the middle. This type of sup-tRNA has recently been artificially evolved to develop biosensors. In these biosensors, an analyte induces the processing of an engineered premature sup-tRNA into a mature sup-tRNA, which suppresses the corresponding nonsense codon incorporated into a gene, encoding an easily detectable reporter protein. This review introduces sup-tRNA-based biosensors that the author's group has developed by utilizing bacterial and eukaryotic cell-free translation systems.

Protein secondary structure determines the temporal relationship between folding and disulfide formation

How and when disulfide bonds form in proteins relative to the stage of their folding is a fundamental question in cell biology. Two models describe this relationship: the folded precursor model, in which a nascent structure forms before disulfides do, and the quasi-stochastic model, where disulfides form prior to folding. Here we investigated oxidative folding of three structurally diverse substrates, β2-microglobulin, prolactin, and the disintegrin domain of ADAM metallopeptidase domain 10 (ADAM10), to understand how these mechanisms apply in a cellular context. We used a eukaryotic cell-free translation system in which we could identify disulfide isomers in stalled translation intermediates to characterize the timing of disulfide formation relative to translocation into the endoplasmic reticulum and the presence of non-native disulfides. Our results indicate that in a domain lacking secondary structure, disulfides form before conformational folding through a process prone to nonnative disulfide formation, whereas in proteins with defined secondary structure, native disulfide formation occurs after partial folding. These findings reveal that the nascent protein structure promotes correct disulfide formation during cotranslational folding.

Mutation of the start codon to enhance Cripavirus internal ribosome entry site-mediated translation in a wheat germ extract.

Wheat germ extract (WGE) is one of the most widely used eukaryotic cell-free translation systems for easy synthesis of a broad range of proteins merely by adding template mRNAs. Its productivity has thus far been improved by removing translational inhibitors from the extract and stabilizing the template with terminal protectors. Nonetheless, there remains room for increasing the yield by designing a terminally protected template with higher susceptibility to translation. Given the fact that a 5' terminal protector is a strong inhibitor of the canonical translation, we herein focused on Cripavirus internal ribosome entry sites (IRESes), which allow for a unique translation initiation from a non-AUG start codon without the help of any initiation factors. We mutated their start codons to enhance the IRES-mediated translation efficiency in WGE. One of the mutants showed considerably higher efficiency, 3-4-fold higher than that of its wild type, and also 3-4-fold higher than the canonical translation efficiency by an IRES-free mRNA having one of the most effective canonical-translation enhancers. Because this mutated IRES is compatible with different types of genes and terminal protectors, we expect it will be widely used to synthesize proteins in WGE.

Artificial OFF-Riboswitches That Downregulate Internal Ribosome Entry without Hybridization Switches in a Eukaryotic Cell-Free Translation System.

We constructed novel artificial riboswitches that function in a eukaryotic translation system (wheat germ extract), by rationally implanting an in vitro-selected aptamer into the intergenic internal ribosome entry site (IRES) of Plautia stali intestine virus. These eukaryotic OFF-riboswitches (OFF-eRSs) ligand-dose-dependently downregulate IRES-mediated translation without hybridization switches, which typical riboswitches utilize for gene regulation. The hybridization-switch-free mechanism not only allows for easy design but also requires less energy for regulation, resulting in a higher switching efficiency than hybridization-switch-based OFF-eRSs provide. In addition, even a small ligand such as theophylline can induce satisfactory repression, in contrast to other types of OFF-eRSs that modulate the 5' cap-dependent canonical translation. Because our proposed hybridization-switch-free OFF-eRSs are based on a versatile IRES that functions well in many types of eukaryotic translation systems, they would be widely usable elements for synthetic gene circuits in both cell-free and cell-based synthetic biology.

Inter-polysomal coupling of termination and initiation during translation in eukaryotic cell-free system.

The recording of the luciferase-generated luminescence in the eukaryotic cell-free translation system programmed with mRNA encoding firefly luciferase (Luc-mRNA) showed that the addition of free exogenous mRNAs into the translation reactor induces the immediate release of the functionally active protein from the polyribosomes of the translation system. The phenomenon did not depend on the coding specificity of the added free mRNA. At the same time it depended on the “initiation potential” of the added mRNA (including the features that ensure the successful initiation of translation, such as the presence of the cap structure and the sufficient concentration of the added mRNA in the translation mixture). The phenomenon also strictly depended on the presence of the stop codon in the translated mRNA. As the above-mentioned features of the added mRNA imply its activity in initiation of a new translation, the experimental data are found in agreement with the scenario where the molecules of the added mRNA interact by their 5′-ends with terminating and recycling ribosomes, stimulating the release of the complete polypeptides and providing for the initiation of a new translation.

Automated production of functional membrane proteins using eukaryotic cell-free translation systems

Due to their high abundance and pharmacological relevance there is a growing demand for the efficient production of functional membrane proteins. In this context, cell-free protein synthesis represents a valuable alternative that allows for the high-throughput synthesis of functional membrane proteins. Here, we demonstrate the potential of our cell-free protein synthesis system, based on lysates from cultured Spodoptera frugiperda 21 cells, to produce pro- and eukaryotic membrane proteins with individual topological characteristics in an automated fashion. Analytical techniques, including confocal laser scanning microscopy, fluorescence detection of eYFP fusion proteins in a microplate reader and in-gel fluorescence of statistically incorporated fluorescent amino acid derivatives were employed. The reproducibility of our automated synthesis approach is underlined by coefficients of variation below 7.2%. Moreover, the functionality of the cell-free synthesized potassium channel KcsA was analyzed electrophysiologically. Finally, we expanded our cell-free membrane protein synthesis system by an orthogonal tRNA/synthetase pair for the site-directed incorporation of p-Azido-l-phenylalanine based on stop codon suppression. Incorporation was optimized by performance of a two-dimensional screening with different Mg2+ and lysate concentrations. Subsequently, the selective modification of membrane proteins with incorporated p-Azido-l-phenylalanine was exemplified by Staudinger ligation with a phosphine-based fluorescence dye.

A continuous-exchange cell-free protein synthesis system based on extracts from cultured insect cells.

In this study, we present a novel technique for the synthesis of complex prokaryotic and eukaryotic proteins by using a continuous-exchange cell-free (CECF) protein synthesis system based on extracts from cultured insect cells. Our approach consists of two basic elements: First, protein synthesis is performed in insect cell lysates which harbor endogenous microsomal vesicles, enabling a translocation of de novo synthesized target proteins into the lumen of the insect vesicles or, in the case of membrane proteins, their embedding into a natural membrane scaffold. Second, cell-free reactions are performed in a two chamber dialysis device for 48 h. The combination of the eukaryotic cell-free translation system based on insect cell extracts and the CECF translation system results in significantly prolonged reaction life times and increased protein yields compared to conventional batch reactions. In this context, we demonstrate the synthesis of various representative model proteins, among them cytosolic proteins, pharmacological relevant membrane proteins and glycosylated proteins in an endotoxin-free environment. Furthermore, the cell-free system used in this study is well-suited for the synthesis of biologically active tissue-type-plasminogen activator, a complex eukaryotic protein harboring multiple disulfide bonds.

Membrane assembly of the functional KcsA potassium channel in a vesicle-based eukaryotic cell-free translation system

The potassium channel KcsA was heterologously expressed in a eukaryotic cell-free system. Both, the expression yields and functional analysis of the protein were reported. Qualitative and quantitative analyses of KcsA expression were performed by using 14C-labeled leucine as one of the amino acids supplemented in the cell-free reaction mixture. There was a time dependent increase in the protein yield as well as the intensity of the native tetramer band in insect cell derived microsomes. Electrophysiology measurements demonstrated the functional activity of the microsomes harboring KcsA showing single-channel currents with the typical biophysical characteristics of the ion channel. The channel behavior was asymmetric and showed positive rectification with larger currents towards positive voltages. KcsA channel currents were effectively blocked by potassium selective barium (Ba2+). This functional demonstration of an ion channel in eukaryotic cell-free system has a large potential for future applications including drug screening, diagnostic applications and functional assessment of complex membrane proteins like GPCRs by coupling them to ion channels in cell-free systems. Furthermore, membrane proteins can be expressed directly from linear DNA templates within 90min, eliminating the need for additional cloning steps, which makes this cell-free system fast and efficient.

Synthesis and site-directed fluorescence labeling of azido proteins using eukaryotic cell-free orthogonal translation systems

Eukaryotic cell-free systems based on wheat germ and Spodoptera frugiperda insect cells were equipped with an orthogonal amber suppressor tRNA–synthetase pair to synthesize proteins with a site-specifically incorporated p-azido-l-phenylalanine residue in order to provide their chemoselective fluorescence labeling with azide-reactive dyes by Staudinger ligation. The specificity of incorporation and bioorthogonality of labeling within complex reaction mixtures was shown by means of translation and fluorescence detection of two model proteins: β-glucuronidase and erythropoietin. The latter contained the azido amino acid in proximity to a signal peptide for membrane translocation into endogenous microsomal vesicles of the insect cell-based system. The results indicate a stoichiometric incorporation of the azido amino acid at the desired position within the proteins. Moreover, the compatibility of cotranslational protein translocation, including glycosylation and amber suppression-based incorporation of p-azido-l-phenylalanine within a cell-free system, is demonstrated. The presented approach should be particularly useful for providing eukaryotic and membrane-associated proteins for investigation by fluorescence-based techniques.

Production of functional antibody fragments in a vesicle-based eukaryotic cell-free translation system

Cell-free protein synthesis is of increasing interest for the rapid and high-throughput synthesis of many proteins, in particular also antibody fragments. In this study, we present a novel strategy for the production of single chain antibody fragments (scFv) in a eukaryotic in vitro translation system. This strategy comprises the cell-free expression, isolation and label-free interaction analysis of a model antibody fragment synthesized in two differently prepared insect cell lysates. These lysates contain translocationally active microsomal structures derived from the endoplasmic reticulum (ER), allowing for posttranslational modifications of cell-free synthesized proteins. Both types of these insect cell lysates enable the synthesis and translocation of scFv into ER-derived vesicles. However, only the one that has a specifically adapted redox potential yields functional active antibody fragments. We have developed a new methodology for the isolation of functional target proteins based on the translocation of cell-free produced scFv into microsomal structures and subsequent collection of protein-enriched vesicles. Antibody fragments that have been released from these vesicles are shown to be well suited for label-free binding studies. Altogether, these results show the potential of insect cell lysates for the production, purification and selection of antibody fragments in an easy-to-handle and time-saving manner.

Rational design of artificial riboswitches based on ligand-dependent modulation of internal ribosome entry in wheat germ extract and their applications as label-free biosensors

Riboswitches are RNA elements in mRNA that control gene expression in cis in response to their specific ligands. Because artificial riboswitches make it possible to regulate any gene with an arbitrary molecule, they are expected to function as biosensors, in which the output is easily detectable protein expression. I report herein a fully rational design strategy for artificially constructing novel riboswitches that work in a eukaryotic cell-free translation system (wheat germ extract). In these riboswitches, translation mediated by an internal ribosome entry site (IRES) is promoted only in the presence of a specific ligand (ON), while it is inhibited in the absence of the ligand (OFF). The first rationally designed riboswitch, which is regulated by theophylline, showed a high switching efficiency and dependency on theophylline. In addition, based on the design of the theophylline-dependent riboswitch, other three kinds of riboswitches controlled by FMN, tetracycline, and sulforhodamine B, were constructed only by calculating the ΔG value of one stem-loop structure. The rational design strategy described herein is therefore useful for easily producing various ligand-dependent riboswitches, which are available as biosensors for detecting their ligands.

Binding and Action of CEM-101, a New Fluoroketolide Antibiotic That Inhibits Protein Synthesis

We characterized the mechanism of action and the drug-binding site of a novel ketolide, CEM-101, which belongs to the latest class of macrolide antibiotics. CEM-101 shows high affinity for the ribosomes of Gram-negative (Escherichia coli) and Gram-positive (Staphylococcus aureus) bacteria. The ketolide shows high selectivity in its inhibitory action and readily interferes with synthesis of a reporter protein in the bacterial but not eukaryotic cell-free translation system. Binding of CEM-101 to its ribosomal target site was characterized biochemically and by X-ray crystallography. The X-ray structure of CEM-101 in complex with the E. coli ribosome shows that the drug binds in the major macrolide site in the upper part of the ribosomal exit tunnel. The lactone ring of the drug forms hydrophobic interactions with the walls of the tunnel, the desosamine sugar projects toward the peptidyl transferase center and interacts with the A2058/A2509 cleft, and the extended alkyl-aryl arm of the drug is oriented down the tunnel and makes contact with a base pair formed by A752 and U2609 of the 23S rRNA. The position of the CEM-101 alkyl-aryl extended arm differs from that reported for the side chain of the ketolide telithromycin complexed with either bacterial (Deinococcus radiodurans) or archaeal (Haloarcula marismortui) large ribosomal subunits but closely matches the position of the side chain of telithromycin complexed to the E. coli ribosome. A difference in the chemical structure of the side chain of CEM-101 in comparison with the side chain of telithromycin and the presence of the fluorine atom at position 2 of the lactone ring likely account for the superior activity of CEM-101. The results of chemical probing suggest that the orientation of the CEM-101 extended side chain observed in the E. coli ribosome closely resembles its placement in Staphylococcus aureus ribosomes and thus likely accurately reflects interaction of CEM-101 with the ribosomes of the pathogenic bacterial targets of the drug. Chemical probing further demonstrated weak binding of CEM-101, but not of erythromycin, to the ribosome dimethylated at A2058 by the action of Erm methyltransferase.

Aptazyme-based biosensors using a eukaryotic cell-free translation system

I have constructed a novel aptazyme-based biosensor system for detecting cofactors of the aptazymes using a cell-free luciferase synthesis in wheat germ extract. In this system, the activity of the aptazyme that is fused to a 5'-untranslated region of a luciferase gene can be detected as luciferase expression. In translating the aptazyme-fused mRNA as-is using a wheat germ cell-free translation system, the luciferase is almost not expressed because of the following triple suppression effects: (1) 5'-terminal three bases and (2) 5'-terminal duplex prevent the ribosome from binding to own mRNA; (3) if the ribosome binds, translation of a mimic gene in the aptazyme inhibits that of the downstream luciferase gene (OFF state). In contrast, in the presence of the aptazyme cofactor, the aptazyme in mRNA is self-cleaved to produce an aptazyme-free luciferase gene, which is translated efficiently (ON state). The ON/OFF efficiency and the detection limit of the aptazyme-based biosensor for theophylline are much higher and lower, respectively, compared to those of previously-reported one that utilizes a prokaryotic translation system.

Protein Methyltransferase Activities in Commercial In vitro Translation Systems

Protein arginine methylation is a well-known post-translational modification that has been shown to occur in rabbit reticulocyte in vitro translation lysates (RRL); however, it is not known whether this is a general feature of in vitro-produced proteins from other eukaryotic cell-free translation systems, particularly insect-derived lysates (ICL). Because methylation can affect protein localization, RNA binding and protein-protein interactions this may be of great importance as in vitro-produced proteins are often used in assays of protein function. Here, I report the presence of base-stable and base-labile methyltransferase activities in RRL, ICL and wheat germ in vitro extracts (WGE). Indeed, the presence of CARM1 in RRL and ICL and a class II protein arginine methyltransferase activity (PRMT5/7) is documented in all three systems. Additionally, the lysine methyltransferase that modifies eukaryotic elongation factor 1A (eEF-1A) was detected in ICL and WGE. Importantly, using a defined set of substrates under identical conditions I show that all three in vitro systems contain different complements of the various methyltransferases. These data suggest that three systems can be used in a complementary fashion to investigate the effect(s) of post-translational modification on protein function.

Ribosome can resume the translation in both + 1 or − 1 frames after encountering an AGA cluster in Escherichia coli

In Escherichia coli the rare codons AGG, AGA and CGA are reported to have a detrimental effect on protein synthesis, especially during the expression of heterologous proteins. In the present work, we have studied the impact of successive clusters of these rare codons on the accuracy of mRNA translation in E. coli. For this purpose, we have analyzed the expression of an mRNA which contains in its 3′ region a triplet and a tandem of AGA codons. This mRNA is derived from the human hepatitis B virus (HBV) preC gene. Both in eukaryotic cells and in eukaryotic cell-free translation system, this mRNA, directs the synthesis of a single 25 kDa protein. However, in a conventional E. coli strain BL 21 (DE3), transformed with a plasmid expressing this protein the synthesis of four polypeptides ranging from 30 to 21.5 kDa can be observed. Using different approaches, notably expression of i) precore mutated proteins or ii) chimeric proteins containing HA- and Myc-tags downstream of the AGA clusters (respectively in the − 1 or + 1 frame), we have found that when the ribosome encounters the AGA clusters, it can then resume the translation in both + 1 and − 1 frames. This result is in agreement with the model proposed recently by Baranov et al. (Baranov, P.V., Gesteland, R.F., Atkins, J.F., 2004. P-site tRNA is a crucial initiator of ribosomal frameshifting. RNA 10, 221–230), thus confirming that AGA/AGG codons can serve as sites for − 1 frameshifting events. Only + 1 frameshifting was suggested previously to occur at the AGA/AGG clusters.

5′‐poly(A) sequence as an effective leader for translation in eukaryotic cell‐free systems

Poly(A) sequence of 25 adenylic residues placed immediately before the start codons of the green fluorescent protein (GFP) and firefly luciferase (Luc) mRNAs is shown to provide a high rate of translation of the heterologous messages in eukaryotic cell-free translation systems. Also the poly(A) leader is found to provide the abolition of the inhibition of translation at excess mRNA concentrations. The possibility of the practical use of the constructs with the poly(A) leader for preparative protein production is demonstrated in the wheat germ continuous-exchange cell-free (CECF) translation system.

Four-base codon-mediated incorporation of nonnatural amino acids into proteins in a eukaryotic cell-free translation system

Various four-base codons have been shown to work for the introduction of non-natural amino acids into proteins in an Escherichia coli cell-free translation system. Here, a four-base codon-mediated non-natural mutagenesis was applied to a eukaryotic rabbit reticulocyte cell-free translation system. Mutated streptavidin mRNAs containing four-base codons were prepared and added to a rabbit reticulocyte lysate in the presence of tRNAs that were aminoacylated with a non-natural amino acid and had the corresponding four-base anticodons. A Western blot analysis of translation products indicated that the four-base codons CGGU, CGCU, CCCU, CUCU, CUAU, and GGGU were efficiently decoded by the aminoacyl-tRNAs having the corresponding four-base anticodons. In contrast, the four-base codons AGGU, AGAU, CGAU, UUGU, UCGU, and ACGU were not decoded. The stop codon-derived four-base codons UAGU, UAAU, and UGAU were found to be inefficient, whereas the amber codon UAG and opal codon UGA were efficient for the incorporation of non-natural amino acids. The application of the expanded genetic code in a eukaryotic cell-free system opens the possibility of a four-base codon-mediated incorporation of non-natural amino acids into proteins in living eukaryotic cells.

Cell-free protein synthesis systems: increasing their performance and applications.

The Escherichia coli cell-free protein synthesis system can now be used for various proteins that need special requirements, such as disulfide bond formation between intra- and inter-molecules, hetero-dimerization, and specific chaperons. In addition, a novel protein library construction method termed "SIMPLEX" has been developed. Some applications with SIMPLEX are described. A highly efficient eukaryotic cell-free translation system using wheat germ extract has also been developed. An advanced framework for a rapid eukaryotic gene expression and analysis is shown.

Cell-free expression and functional reconstitution of CALM in clathrin assembly.

Clathrin-mediated vesicle formation is an essential step in the intracellular trafficking of the protein and lipid. Binding of clathrin assembly protein to clathrin triskelia induces their assembly into clathrin-coated vesicles (CCVs). In order to better understand a possible role of post-translational modification of CALM (clathrin assembly protein lymphoid myeloid), the homologue of AP180, in the assembly of CCVs, CALM was expressed in the cell-free reticulocyte translation system that is capable of carrying out post-translational modification. The apparent molecular weight of the expressed recombinant CALM was estimated as 105 kD. Alkaline phosphatase treatment of CALM resulted in a mobility shift on SDS-PAGE. We found that CALM was associated with the proteins harboring SH3 domain, promote assembly of clathrin triskelia into clathrin cage and bound to the preformed clathrin cage. CALM was also proteolyzed by caspase 3 and calpain but not by caspase 8. These results indicated that the post-translationally modified CALM, expressed in the eukaryotic cell-free reticulocyte translation system was able to mediate the assembly of clathrin and the coated-vesicle formation.