Cell-free Translation System Research Articles

Consecutive Elongation of D-Amino Acids in Translation

Recent progress in the field of genetic code reprogramming using a reconstituted cell-free translation system has made it possible to incorporate a wide array of non-proteinogenic amino acids, including N-methyl-amino acids and D-amino acids. Despite the fact that up to ten N-methyl-amino acid residues can be continuously elongated, the successive incorporation of even two D-amino acids into a nascent peptide chain remains a formidable challenge, thus far being nearly impossible. Here we report achievement of continuous D-amino acid elongation by the use of engineered tRNAs and optimized concentrations of translation factors, enabling us to incorporate up to ten consecutive D-Ser residues into a nascent peptide chain. We have also expressed macrocyclic peptides consisting of four or five consecutive D-amino acids consisting of D-Phe, D-Ser, D-Ala, or D-Cys closed by either a disulfide bond or a thioether bond.

The Minimum Open Reading Frame, AUG-Stop, Induces Boron-Dependent Ribosome Stalling and mRNA Degradation.

Upstream open reading frames (uORFs) are often translated ahead of the main ORF of a gene and regulate gene expression, sometimes in a condition-dependent manner, but such a role for the minimum uORF (hereafter referred to as AUG-stop) in living organisms is currently unclear. Here, we show that AUG-stop plays an important role in the boron (B)-dependent regulation of NIP5;1, encoding a boric acid channel required for normal growth under low B conditions in Arabidopsis thaliana High B enhanced ribosome stalling at AUG-stop, which was accompanied by the suppression of translation and mRNA degradation. This mRNA degradation was promoted by an upstream conserved sequence present near the 5'-edge of the stalled ribosome. Once ribosomes translate a uORF, reinitiation of translation must take place in order for the downstream ORF to be translated. Our results suggest that reinitiation of translation at the downstream NIP5;1 ORF is enhanced under low B conditions. A genome-wide analysis identified two additional B-responsive genes, SKU5 and the transcription factor gene ABS/NGAL1, which were regulated by B-dependent ribosome stalling through AUG-stop. This regulation was reproduced in both plant and animal transient expression and cell-free translation systems. These findings suggest that B-dependent AUG-stop-mediated regulation is common in eukaryotes.

Upstream Open Reading Frames Located in the Leader of Protein Kinase Mζ mRNA Regulate Its Translation.

For protein synthesis that occurs locally in dendrites, the translational control mechanisms are much more important for neuronal functioning than the transcription levels. Here, we show that uORFs (upstream open reading frames) in the 5′ untranslated region (5′UTR) play a critical role in regulation of the translation of protein kinase Mζ (PKMζ). Elimination of these uORFs activates translation of the reporter protein in vitro and in primary cultures of rat hippocampal neurons. Using cell-free translation systems, we demonstrate that translational initiation complexes are formed only on uORFs. Further, we address the mechanism of translational repression of PKMζ translation, by uORFs. We observed an increase in translation of the reporter protein under the control of PKMζ leader in neuronal culture during non-specific activation by picrotoxin. We also show that such a mechanism is similar to the mechanism seen in cell stress, as application of sodium arsenite to neuron cultures induced translation of mRNA carrying PKMζ 5′UTR similarly to picrotoxin activation. Therefore, we suppose that phosphorylation of eIF2a, like in cell stress, is a main regulator of PKMζ translation. Altogether, our findings considerably extend our understanding of the role of uORF in regulation of PKMζ translation in activated neurons, important at early stages of LTP.

Cell-free analysis of polyQ-dependent protein aggregation and its inhibition by chaperone proteins

Protein misfolding and aggregation is one of the major causes of neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease and Huntington's disease. So far protein aggregation related to these diseases has been studied using animals, cultured cells or purified proteins. In this study, we show that a newly synthesized polyglutamine protein implicated in Huntington's disease forms large aggregates in HeLa cells, and successfully recapitulate the process of this aggregation using a translation-based system derived from HeLa cell extracts. When the cell-free translation system was pre-incubated with recombinant human cytosolic chaperonin CCT, or the Hsc70 chaperone system (Hsc70s: Hsc70, Hsp40, and Hsp110), aggregate formation was inhibited in a dose-dependent manner. In contrast, when these chaperone proteins were added in a post-translational manner, aggregation was not prevented. These data led us to suggest that chaperonin CCT and Hsc70s interact with nascent polyglutamine proteins co-translationally or immediately after their synthesis in a fashion that prevents intra- and intermolecular interactions of aggregation-prone polyglutamine proteins. We conclude that the in vitro approach described here can be usefully employed to analyze the mechanisms that provoke polyglutamine-driven protein aggregation and to screen for molecules to prevent it.

Chimeric RNA Oligonucleotides Incorporating Triazole-Linked Trinucleotides: Synthesis and Function as mRNA in Cell-Free Translation Reactions.

A method for the synthesis of chimeric oligonucleotides was developed to incorporate purine nucleobases and multiple triazole linkers in natural, phosphate-linked structures of RNA. A solution-phase synthesis method for triazole-linked RNA oligomers via copper-catalyzed azide-alkyne cycloaddition reaction was optimized and tolerated purine nucleobases and protecting groups for further transformations. Three TLRNA trinucleotides with 5'-protected hydroxy and 3'-phosphoramidite groups were prepared, and one congener with a representative sequence was subjected to automated, solid-phase phosphoramidite synthesis. The synthesis allowed the efficient preparation of 13-mer chimeric RNA oligonucleotides with two triazole linkers, ten phosphate linkers and purine/pyrimidine nucleobases. The chimeric oligonucleotide was found applicable to a cell-free translation system as mRNA and provided the genetic code for dipeptide production.

Cell-free synthesis of functional human epidermal growth factor receptor: Investigation of ligand-independent dimerization in Sf21 microsomal membranes using non-canonical amino acids

Cell-free protein synthesis systems represent versatile tools for the synthesis and modification of human membrane proteins. In particular, eukaryotic cell-free systems provide a promising platform for their structural and functional characterization. Here, we present the cell-free synthesis of functional human epidermal growth factor receptor and its vIII deletion mutant in a microsome-containing system derived from cultured Sf21 cells. We provide evidence for embedment of cell-free synthesized receptors into microsomal membranes and asparagine-linked glycosylation. Using the cricket paralysis virus internal ribosome entry site and a repetitive synthesis approach enrichment of receptors inside the microsomal fractions was facilitated thereby providing analytical amounts of functional protein. Receptor tyrosine kinase activation was demonstrated by monitoring receptor phosphorylation. Furthermore, an orthogonal cell-free translation system that provides the site-directed incorporation of p-azido-L-phenylalanine is characterized and applied to investigate receptor dimerization in the absence of a ligand by photo-affinity cross-linking. Finally, incorporated azides are used to generate stable covalently linked receptor dimers by strain-promoted cycloaddition using a novel linker system.

Cell-Free Technologies for Proteomics and Protein Engineering.

Cell-free translation systems facilitate rapid production of specific proteins and are particularly suited as high-throughput methods for whole-genome protein synthesis. Moreover, these systems do not rely on living cells, thereby allowing the synthesis of unstable or cytotoxic proteins in vitro. In this review, we describe the principles and potential applications of cell-free protein translation systems and the future prospects of proteomics approaches using next-generation sequencing and cell-free expression technologies.

High-yield cell-free synthesis of human EGFR by IRES-mediated protein translation in a continuous exchange cell-free reaction format

Cell-free protein synthesis systems derived from eukaryotic sources often provide comparatively low amounts of several μg per ml of de novo synthesized membrane protein. In order to overcome this, we herein demonstrate the high-yield cell-free synthesis of the human EGFR in a microsome-containing system derived from cultured Sf21 cells. Yields were increased more than 100-fold to more than 285 μg/ml by combination of IRES-mediated protein translation with a continuous exchange cell-free reaction format that allowed for prolonged reaction lifetimes exceeding 24 hours. In addition, an orthogonal cell-free translation system is presented that enabled the site-directed incorporation of p-Azido-L-phenylalanine by amber suppression. Functionality of cell-free synthesized receptor molecules is demonstrated by investigation of autophosphorylation activity in the absence of ligand and interaction with the cell-free synthesized adapter molecule Grb2.

A reconstituted cell-free assay for the evaluation of the intrinsic activity of purified human ribosomes.

We describe a cell-free translation system for evaluating the activity of ribosomes stringently purified from human cells. This system is based on in vitro reconstitution of the cellular translation machinery, in which a ribosome-free rabbit reticulocyte lysate (RRL) is reassembled with human ribosomes and in vitro-transcribed reporter mRNAs. The protocol describes the preparation of the RRL-derived fractions, purification of ribosomes devoid of detectable nonribosomal-associated factors, and assembly of the reactions to evaluate ribosomal translational efficiency and fidelity using appropriate reporter transcripts. The whole procedure can be completed in ∼2.5 d (plus 2 weeks for RRL preparation and cell expansion time). This protocol can be applied to study intrinsic functional properties (cis-acting element-mediated translation initiation or translational fidelity) of ribosome populations from different sources (including nonhuman origin). It is therefore useful for the characterization of ribosomal function in ribosomopathies and cancer, and it will be applicable in the emerging fields of ribosome diversity and specialized ribosomes.

Effect of Liposome Size on Internal RNA Replication Coupled with Replicase Translation.

Cell membranes inhibit the diffusion of intracellular materials, and compartment size can strongly affect the intracellular biochemical reactions. To assess the effect of the size of microcompartments on intracellular reactions, we constructed a primitive cell model consisting of giant liposomes and a translation-coupled RNA replication (TcRR) system. The RNA was replicated by Qβ replicase, which was translated from the RNA in giant liposomes encapsulating the cell-free translation system. A reporter RNA encoding the antisense strand of β-glucuronidase was introduced into the system to yield a TcRR read-out (green fluorescence). We demonstrate that TcRR was hardly detectable in larger liposomes (230 fL) but was more effective in smaller (7.7 fL) liposomes. Our experimental and theoretical results show that smaller microcompartments considerably enhance TcRR because the synthesized molecules, such as RNA and replicases, are more concentrated in smaller liposomes.

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.

AmyI-1–18, a cationic α-helical antimicrobial octadecapeptide derived from α-amylase in rice, inhibits the translation and folding processes in a protein synthesis system

In our previous study, we used a cell-free rapid translation system (RTS), which is an invitro protein synthesis system based on Escherichia coli lysate, for evaluating the inhibition of green fluorescent protein (GFP) synthesis by pyrrhocoricin. In this study, using an RTS, we evaluated the inhibition of GFP synthesis by AmyI-1-18, an antimicrobial octadecapeptide. We found that, similarly to pyrrhocoricin, AmyI-1-18 inhibited GFP synthesis in the RTS in a concentration-dependent manner. In addition, the blockage of transcription and translation steps in the RTS was individually estimated using RT-PCR after gene expression to determine the mRNA products and using sodium dodecyl sulfate-polyacrylamide gel electrophoresis to determine the amounts of GFP expressed from purified mRNA, respectively. The results demonstrated that the inhibition of GFP synthesis by AmyI-1-18 did not occur at the transcription step but rather at the translation step. Furthermore, we assessed the inhibition of DnaK-mediated refolding of chemically denatured luciferase by AmyI-1-18; AmyI-1-18 inhibited the protein folding activity of the ATP-dependent DnaK/DnaJ molecular chaperone system in a concentration-dependent manner. Surface plasmon resonance (SPR) analysis showed that AmyI-1-18 strongly bound to RNA with a KD value of 1.4×10(-8)M but not to DNA and that AmyI-1-18 specifically bound to DnaK with a KD value of 4.4×10(-6)M. These SPR analysis results supported the results obtained in both the RTS and the molecular chaperone system. These results demonstrated that both RNA and DnaK are most likely the target of AmyI-1-18 in the protein synthesis system.

Directed Evolution of a Cyclized Peptoid-Peptide Chimera against a Cell-Free Expressed Protein and Proteomic Profiling of the Interacting Proteins to Create a Protein-Protein Interaction Inhibitor.

N-alkyl amino acids are useful building blocks for the in vitro display evolution of ribosomally synthesized peptides because they can increase the proteolytic stability and cell permeability of these peptides. However, the translation initiation substrate specificity of nonproteinogenic N-alkyl amino acids has not been investigated. In this study, we screened various N-alkyl amino acids and nonamino carboxylic acids for translation initiation with an Escherichia coli reconstituted cell-free translation system (PURE system) and identified those that efficiently initiated translation. Using seven of these efficiently initiating acids, we next performed in vitro display evolution of cyclized peptidomimetics against an arbitrarily chosen model human protein (β-catenin) cell-free expressed from its cloned cDNA (HUPEX) and identified a novel β-catenin-binding cyclized peptoid-peptide chimera. Furthermore, by a proteomic approach using direct nanoflow liquid chromatography-tandem mass spectrometry (DNLC-MS/MS), we successfully identified which protein-β-catenin interaction is inhibited by the chimera. The combination of in vitro display evolution of cyclized N-alkyl peptidomimetics and in vitro expression of human proteins would be a powerful approach for the high-speed discovery of diverse human protein-targeted cyclized N-alkyl peptidomimetics.

Ribosomal Synthesis of Peptides with Multiple β-Amino Acids.

The compatibility of β-amino acids with ribosomal translation was studied for decades, but it has been still unclear whether the ribosome can accept various β-amino acids, and whether the ribosome can introduce multiple β-amino acids in a peptide. In the present study, by using the Escherichia coli reconstituted cell-free translation system with a reprogramed genetic code, we screened β-amino acids that give high single incorporation efficiency and used them to synthesize peptides containing multiple β-amino acids. The experiments of single β-amino acid incorporation into a peptide revealed that 13 β-amino acids are compatible with ribosomal translation. Six of the tested β-amino acids (βhGly, l-βhAla, l-βhGln, l-βhPhg, l-βhMet, and d-βhPhg) showed high incorporation efficiencies, and seven (l-βhLeu, l-βhIle, l-βhAsn, l-βhPhe, l-βhLys, d-βhAla, and d-βhLeu) showed moderate incorporation efficiencies; whereas no full-length peptide was produced using other β-amino acids (l-βhPro, l-βhTrp, and l-βhGlu). Subsequent double-incorporation experiments using β-amino acids with high single incorporation efficiency revealed that elongation of peptides with successive β-amino acids is prohibited. Efficiency of the double-incorporation of the β-amino acids was restored by the insertion of Tyr or Ile between the two β-amino acids. On the basis of these experiments, we also designed mRNA sequences of peptides, and demonstrated the ribosomal synthesis of peptides containing different types of β-amino acids at multiple positions.

Double-Fluorescent-Labeled Single-Chain Antibodies Showing Antigen-Dependent Fluorescence Ratio Change

Abstract Fluorescence ratio probes are useful tools for quantitative detection of target molecules even if the concentration of probe molecules is unknown. However, a general and widely applicable method for design and synthesis of fluorescence ratio probes has never been established. Here, we developed double-labeled single-chain antibody fragment (scFv) derivatives showing antigen-dependent fluorescence ratio change based on fluorescence resonance energy transfer (FRET) and antigen-dependent fluorescence quenching. Double-labeled scFvs were synthesized by incorporating fluorescent nonnatural amino acids labeled with TAMRA and Rhodamine Green (RhG) as a FRET donor and acceptor pair into N- and C-termini of scFv, respectively, using four-base and amber codons in a cell-free translation system. The resulting double-labeled antibody fragments showed significant fluorescence ratio change upon the antigen-binding. This result was explained by FRET occurring from RhG to TAMRA but TAMRA being quenched by Trp residues in the absence of antigen. The binding of the antigen canceled the quenching of TAMRA without changing FRET efficiency. Double-labeled antibody fragments developed here will be useful as diagnostic and imaging tools.

Inhibition of Mitogen-activated Protein Kinase (MAPK)-interacting Kinase (MNK) Preferentially Affects Translation of mRNAs Containing Both a 5′-Terminal Cap and Hairpin

The MAPK-interacting kinases 1 and 2 (MNK1 and MNK2) are activated by extracellular signal-regulated kinases 1 and 2 (ERK1/2) or p38 in response to cellular stress and extracellular stimuli that include growth factors, cytokines, and hormones. Modulation of MNK activity affects translation of mRNAs involved in the cell cycle, cancer progression, and cell survival. However, the mechanism by which MNK selectively affects translation of these mRNAs is not understood. MNK binds eukaryotic translation initiation factor 4G (eIF4G) and phosphorylates the cap-binding protein eIF4E. Using a cell-free translation system from rabbit reticulocytes programmed with mRNAs containing different 5'-ends, we show that an MNK inhibitor, CGP57380, affects translation of only those mRNAs that contain both a cap and a hairpin in the 5'-UTR. Similarly, a C-terminal fragment of human eIF4G-1, eIF4G(1357-1600), which prevents binding of MNK to intact eIF4G, reduces eIF4E phosphorylation and inhibits translation of only capped and hairpin-containing mRNAs. Analysis of proteins bound to m(7)GTP-Sepharose reveals that both CGP and eIF4G(1357-1600) decrease binding of eIF4E to eIF4G. These data suggest that MNK stimulates translation only of mRNAs containing both a cap and 5'-terminal RNA duplex via eIF4E phosphorylation, thereby enhancing the coupled cap-binding and RNA-unwinding activities of eIF4F.

Expanding the amino acid repertoire of ribosomal polypeptide synthesis via the artificial division of codon boxes.

In ribosomal polypeptide synthesis the library of amino acid building blocks is limited by the manner in which codons are used. Of the proteinogenic amino acids, 18 are coded for by multiple codons and therefore many of the 61 sense codons can be considered redundant. Here we report a method to reduce the redundancy of codons by artificially dividing codon boxes to create vacant codons that can then be reassigned to non-proteinogenic amino acids and thereby expand the library of genetically encoded amino acids. To achieve this, we reconstituted a cell-free translation system with 32 in vitro transcripts of transfer RNASNN (tRNASNN) (S = G or C), assigning the initiator and 20 elongator amino acids. Reassignment of three redundant codons was achieved by replacing redundant tRNASNNs with tRNASNNs pre-charged with non-proteinogenic amino acids. As a demonstration, we expressed a 32-mer linear peptide that consists of 20 proteinogenic and three non-proteinogenic amino acids, and a 14-mer macrocyclic peptide that contains more than four non-proteinogenic amino acids.

Preparation of Quenchbodies by protein transamination reaction

Quenchbody (Q-body) is an antibody fragment labeled with fluorescent dye(s), which functions as a biosensor via the antigen-dependent removal of the quenching effect on fluorophores. It is based on the principle that the fluorescence of the dye(s) attached to the antibody N-terminal region is quenched primarily by the tryptophan residues present in the variable regions, and this quenching is released when the antigen binds to the antibody, resulting in increased fluorescence intensity. Hence Q-body is utilized in various immunoassays for the rapid and sensitive detection of analytes. So far, Q-bodies have been prepared by using a cell-free translation system or by combining Escherichia coli expression and post-labeling steps. However, the above methods need antibody gene cloning, and are time-consuming. In this study, we report a novel approach to prepare Q-bodies by protein N-terminal transamination. We used the antigen-binding fragment (Fab) of an antibody against the bone-Gla-protein (BGP), a biomarker for bone diseases, which was expressed in E.coli. The purified Fab was treated with Rapoport's salt to convert the amino group at the N-terminus to a ketone group, which in turn was allowed to react with fluorescent probes that have aminooxy or hydrazide groups, to prepare a Q-body. The Q-body prepared by this method could detect the BGP-C7 antigen at concentrations as low as 10nM. Since the approach can label the protein N-terminus directly, it could be applied for preparing Q-bodies from natural antibodies and for the rapid screening of high-performance Q-bodies.

Synthesis and Characterization of ¹⁸F-Interleukin-8 Using a Cell-Free Translation System and 4-¹⁸F-Fluoro-L-Proline.

Macromolecules such as proteins are attracting increasing interest for molecular imaging. We previously proposed a novel strategy for preparing macromolecules labeled with a PET radionuclide, (11)C, using a cell-free translation system with (11)C-methionine. However, macromolecules tend to exhibit slower kinetics, thus requiring a longer scanning time. Here, we expand our strategy using (18)F, which has a longer half-life, with the cell-free translation system with 4-(18)F-fluoro-L-proline ((18)F-FPro). We evaluated (18)F-interleukin-8 ((18)F-IL-8) produced by this method in vitro and in vivo to provide a proof of concept of our strategy. We tested some fluorinated amino acids to be incorporated into a protein. Trans-(18)F-FPro was radiolabeled from the corresponding precursor. (18)F-IL-8 was produced using the cell-free translation system with trans-(18)F-FPro instead of natural L-proline with incubation at 37°C for 120 min. An in vitro binding assay of (18)F-IL-8 was performed using IL-8 receptor-expressing cells. After intravenous administration of (18)F-IL-8, in vivo PET imaging of IL-8 receptor-expressing xenograft-bearing mice was performed using a small-animal PET system. FPro was identified as an amino acid incorporated into the protein. (18)F-IL-8 was successfully prepared using the cell-free translation system and trans-(18)F-FPro with the radiochemical yield of 1.5% (decay-corrected) based on trans-(18)F-FPro. In vitro binding assays of (18)F-IL-8 demonstrated its binding to IL-8 receptor. In vivo PET imaging demonstrated that (18)F-IL-8 clearly accumulated in IL-8 receptor-expressing xenografts in mice, unlike trans-(18)F-FPro. (18)F-IL-8 produced by this method binds to IL-8 receptors in vitro, and (18)F-IL-8 PET clearly visualizes its target receptor-expressing xenograft in vivo. Therefore, this technique might be useful for labeling macromolecules and performing preclinical evaluations of proteins of interest in vitro and in vivo.

Leishmania tarentolae for the Production of Multi-subunit Complexes.

Multi-subunit protein complexes are involved in a wide variety of cellular processes including DNA replication, transcriptional regulation, signal transduction, protein folding and degradation. A better understanding of the function of these protein complexes requires structural insights into the molecular arrangement and interactions of their constituent subunits. However, biochemical and structural analysis of multi-subunit protein complexes is still limited because of technical difficulties with their recombinant expression and reconstitution. This chapter presents an overview of a novel protein expression system based on Leishmania tarentolae, a unicellular protozoan parasite of lizards, and practical considerations for the production of multi-subunit protein complexes. The Leishmania tarentolae expression system offers fully eukaryotic protein expression with post-translational modifications but with ease of handling similar to bacteria. This chapter also summarizes studies on the production of laminins, large heterotrimeric glycoproteins of the extracellular matrix, using this expression system. In addition, a recently developed Leishmania tarentolae-based cell-free translation system is briefly described.