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The wheat germ cell-free protein synthesis system has been used as a eukaryotic protein production system since it was first reported in 1964. Although initially the productivity of this system was not very high, it has now become one of the most versatile protein production systems, thanks to the enhancements made by several groups. In this chapter, we report a protein production method for plant receptor kinases using the wheat cell-free system. We describe a method for the preparation of a cell-free extract from wheat germ, the split-primer PCR method for preparation of transcription templates, and the bilayer cell-free protein synthesis method.

Using a wheat germ cell-free protein synthesis system, we developed a high-throughput method for the synthesis of stable isotope-labeled full-length transmembrane proteins as proteoliposomes to mimic the in vivo environment, and we successfully constructed an internal standard library for targeted transmembrane proteomics by using mass spectrometry.

Whereas the dengue virus (DENV) non-structural (NS) proteins NS3 and NS5 have been shown to interact in vitro and in vivo, the biological relevance of this interaction in viral replication has not been fully clarified. Here, we first applied a simple and robust in vitro assay based on AlphaScreen technology in combination with the wheat-germ cell-free protein production system to detect the DENV-2 NS3-NS5 interaction in a 384-well plate. The cell-free-synthesized NS3 and NS5 recombinant proteins were soluble and in possession of their respective enzymatic activities in vitro. In addition, AlphaScreen assays using the recombinant proteins detected a specific interaction between NS3 and NS5 with a robust Z' factor of 0.71. By employing the AlphaScreen assay, we found that both the N-terminal protease and C-terminal helicase domains of NS3 are required for its association with NS5. Furthermore, a competition assay revealed that the binding of full-length NS3 to NS5 was significantly inhibited by the addition of an excess of NS3 protease or helicase domains. Our results demonstrate that the AlphaScreen assay can be used to discover novel antiviral agents targeting the interactions between DENV NS proteins., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Background: Recently, some groups have reported on cell-free synthesis of functional membrane proteins (MPs) in the presence of exogenous liposomes (liposomes). Previously, we reported synthesis of a functional AtPPT1 plant phosphate transporter that was associated with liposomes during translation. However, it is unclear whether or not lipid/MP complex formation is common to all types of MPs in the wheat cell-free system., Results: AtPPT1 was synthesized using a wheat cell-free system with or without liposomes. AtPPT1 synthesized with liposomes showed high transport activity, but the activity of AtPPT1 synthesized without liposomes was less than 10% activity of that with liposomes. To test whether co-translational association with liposomes is observed in the synthesis of other MPs, we used 40 mammalian MPs having one to 14 transmembrane domains (TMDs) and five soluble proteins as a control. The association rate of all 40 MPs into liposomes was more than 40% (mean value: 59%), while that of the five soluble proteins was less than 20% (mean value: 12%). There were no significant differences in association rate among MPs regardless of the number of TMDs and synthesis yield. These results indicate that the wheat cell-free system is a highly productive method for lipid/MP complex formation and is suitable for large-scale preparation. The liposome association of green fluorescent protein (GFP)-fusion MPs were also tested and recovered as lipid/MP complex after floatation by Accudenz density gradient ultracentrifugation (DGU). Employment of GFP-MPs revealed optimal condition for Accudenz floatation. Using the optimized Accudenz DGU condition, P2RX4/lipid complexes were partially purified and detected as a major band by Coomassie Brilliant Blue (CBB)-staining after SDS-PAGE., Conclusion: Formation of lipid/AtPPT1 complex during the cell-free synthesis reaction is critical for synthesis of a functional MP. The lipid/MP complex during the translation was observed in all 40 MPs tested. At least 29 MPs, as judged by their higher productivity compared to GFP, might be suitable for a large-scale preparation. MPs synthesized by this method form lipid/MP complexes, which could be readily partially purified by Accudenz DGU. Wheat cell-free protein synthesis in the presence of liposomes will be a useful method for preparation of variety type of MPs.

In malaria vaccine research, one of the major obstacles has been the difficulty of expressing recombinant malarial proteins and it is mainly due to the lack of an efficient methodology for the synthesis of sufficient quantity of quality proteins. We demonstrate that the wheat germ cell-free protein synthesis system can be applied for the successful production of leading malaria vaccine candidate antigens and, thus, prove that it may be a key tool for malaria vaccine research.

Background: Ubiquitination is mediated by the sequential action of at least three enzymes: the E1 (ubiquitin-activating enzyme), E2 (ubiquitin-conjugating enzyme) and E3 (ubiquitin ligase) proteins. Polyubiquitination of target proteins is also implicated in several critical cellular processes. Although Arabidopsis genome research has estimated more than 1,300 proteins involved in ubiquitination, little is known about the biochemical functions of these proteins. Here we demonstrate a novel, simple and high-sensitive method for in vitro analysis of ubiquitination and polyubiquitination based on wheat cell-free protein synthesis and luminescent detection., Results: Using wheat cell-free synthesis, 11 E3 proteins from Arabidopsis full-length cDNA templates were produced. These proteins were analyzed either in the translation mixture or purified recombinant protein from the translation mixture. In our luminescent method using FLAG- or His-tagged and biotinylated ubiquitins, the polyubiquitin chain on AtUBC22, UPL5 and UPL7 (HECT) and CIP8 (RING) was detected. Also, binding of ubiquitin to these proteins was detected using biotinylated ubiquitin and FLAG-tagged recombinant protein. Furthermore, screening of the RING 6 subgroup demonstrated that At1g55530 was capable of polyubiquitin chain formation like CIP8. Interestingly, these ubiquitinations were carried out without the addition of exogenous E1 and/or E2 proteins, indicating that these enzymes were endogenous to the wheat cell-free system. The amount of polyubiquitinated proteins in the crude translation reaction mixture was unaffected by treatment with MG132, suggesting that our system does not contain 26S proteasome-dependent protein degradation activity., Conclusion: In this study, we developed a simple wheat cell-free based luminescence method that could be a powerful tool for comprehensive ubiquitination analysis.

Background: The last decade has brought the renaissance of protein studies and accelerated the development of high-throughput methods in all aspects of proteomics. Presently, most protein synthesis systems exploit the capacity of living cells to translate proteins, but their application is limited by several factors. A more flexible alternative protein production method is the cell-free in vitro protein translation. Currently available in vitro translation systems are suitable for high-throughput robotic protein production, fulfilling the requirements of proteomics studies. Wheat germ extract based in vitro translation system is likely the most promising method, since numerous eukaryotic proteins can be cost-efficiently synthesized in their native folded form. Although currently available vectors for wheat embryo in vitro translation systems ensure high productivity, they do not meet the requirements of state-of-the-art proteomics. Target genes have to be inserted using restriction endonucleases and the plasmids do not encode cleavable affinity purification tags., Results: We designed four ligation independent cloning (LIC) vectors for wheat germ extract based in vitro protein translation. In these constructs, the RNA transcription is driven by T7 or SP6 phage polymerase and two TEV protease cleavable affinity tags can be added to aid protein purification. To evaluate our improved vectors, a plant mitogen activated protein kinase was cloned in all four constructs. Purification of this eukaryotic protein kinase demonstrated that all constructs functioned as intended: insertion of PCR fragment by LIC worked efficiently, affinity purification of translated proteins by GST-Sepharose or MagneHis particles resulted in high purity kinase, and the affinity tags could efficiently be removed under different reaction conditions. Furthermore, high in vitro kinase activity testified of proper folding of the purified protein., Conclusion: Four newly designed in vitro translation vectors have been constructed which allow fast and parallel cloning and protein purification, thus representing useful molecular tools for high-throughput production of eukaryotic proteins.

A cell-free protein synthesis system is a powerful tool with which unnatural amino acids can be introduced into polypeptide chains. Here, the authors describe unnatural amino acid probing in a wheat germ cell-free translation system as a method for detecting the structural changes that occur in a cofactor binding protein on a conversion of the protein from an apo-form to a holo-form. The authors selected the FMN-binding protein from Desulfovibrio vulgaris as a model protein. The apo-form of the protein was synthesized efficiently in the absence of FMN. The purified apo-form could be correctly converted to the holo-form. Thus, the system could synthesize the active apo-form. Gel filtration chromatography, analytical ultracentrifugation, and circular dichroism-spectra studies suggested that the FMN-binding site of the apo-form is open as compared with the holo-form. To confirm this idea, the unnatural amino acid probing was performed by incorporating 3-azido-L-tyrosine at the Tyr35 residue in the FMN-binding site. The authors optimized three steps in their system. The introduced 3-azido-L-tyrosine residue was subjected to specific chemical modification by a fluorescein-triarylphosphine derivative. The initial velocity of the apo-form reaction was 20 fold faster than that of the holo-form, demonstrating that the Tyr35 residue in the apo-form is open to solvent., (2007 Wiley-Liss, Inc.)

Among the cell-free protein synthesis systems, the wheat germ-based translation system has significant advantages for the high-throughput production of eukaryotic multidomain proteins in folded state. Here, we describe protocols for this cell-free expression system.

This chapter contains protocols for high-throughput protein production based on the cell-free system prepared from eukaryote wheat embryos.

In the current post-genomic era, cell-free translation platforms are gaining importance in structural as well as functional genomics. They are based on extracts prepared from Escherichia coli cells, wheat germ, or rabbit reticulocytes, and when programmed with any mRNA in the presence of energy sources and amino acids, can synthesize the respective protein in vitro. Among the cell-free systems, the wheat germ-based translation system is of special interest due to its eukaryotic nature and robustness. This chapter outlines the existing protein production platforms and their limitations, and describes the basic concept of the wheat germ-based cell-free system. It also demonstrates how the conventional wheat germ system can be improved by eliminating endogenous inhibitors, by using an expression vector specially designed for this system and polymerase chain reaction-directed protein synthesis directly from cDNAs in a bi-layer translation system. Finally, a robotic procedure for translation based on the wheat germ extract and bi-layer cell-free translation is described.

We report a morphological study of functioning ribosomes in a efficient and robust cell-free protein synthesis system prepared from wheat embryos. Sucrose density gradient analysis of translated mixtures programmed with luciferase mRNAs having different 5' and 3' untranslated regions showed formation of large polysomes. Electron microscopic examination of translation mixtures programmed with those of capped and polyadenylated mRNA revealed that ribosomes assemble into a circular-type polysome in vitro. Furthermore, a series of experiments using mRNAs lacking either cap, poly(A) tail or both also resulted in the formation of circular polysomes, which are indistinguishable from those with the original mRNA. The wheat germ cell-free system may provide a good experimental system for understanding functional ribosomes at the molecular level.

To develop applications of in vitro cell-free translation systems for production and characterization of cofactor binding proteins, we investigate the production of apo- or holo-forms of Flavin Mono Nucleotide (FMN)-binding protein from Desulfovibrio vulgaris (Miyazaki F) and purified them. The redox potential analysis and measurements of UV-, visible, and fluorescent spectra of reconstructed holo-protein showed that the FMN correctly bound to the FMN binding site. On the other hand, contrary to our expectation, we found that the apo-protein formed a dimer structure and the incorporation of the FMN led the conformational alterations of the protein. These studies demonstrate the utility of cell-free translation systems to analyses of cofactor-binding proteins.

We have developed a highly productive cell-free protein synthesis system from wheat germ, which is expected to become an important tool for postgenomic research. However, this system has not been optimized for the synthesis of disulfide-containing proteins. Thus, we searched here for translation conditions under which a model protein, a single-chain antibody variable fragment (scFv), could be synthesized into its active form. Before the start of translation, the reducing agent dithiothreitol, which normally is added to the wheat germ extract but which inhibits disulfide formation during translation, was removed by gel filtration. When the scFv mRNA was incubated with this dithiothreitol-deficient extract, more than half of the synthesized polypeptide was recovered in the soluble fraction. By addition of protein disulfide isomerase in the translation solution, the solubility of the product was further improved, and nearly half of the soluble polypeptides strongly bound to the antigen immobilized on an agarose support. This strong binding component had a high affinity as shown by surface-plasmon resonance analysis. These results show that the wheat germ cell-free system can produce a functional scFv with a simple change of the reaction ingredients. We also discuss protein folding in this system and suggest that the disulfide bridges are formed cotranslationally. Finally, we show that biotinylated scFv could be synthesized in similar fashion and immobilized on a solid surface to which streptavidin is bound. SPR measurements for detection of antigens were also possible with the use of this immobilized surface.

A high-throughput cell-free protein synthesis method has been described. The methodology is based on a bilayer diffusion system that enables the continuous supply of substrates, together with the continuous removal of small byproducts, through a phase between the translation mixture and substrate mixture. With the use of a multititer plate the system was functional for a prolonged time, and as a consequence yielded more than 10 times that of the similar batch-mode reaction. Combining this method with a wheat germ cell-free translation system developed by us, the system could produce a large amount of protein sufficient for carrying out functional analyses. This novel bilayer-based cell-free protein synthesis system with its simplicity, minimum time and low cost may be useful practical methodology in the post-genome era.

Current cell-free protein synthesis systems can synthesize proteins with high speed and accuracy, but produce only a low yield because of their instability over time. Here we describe the preparation of a highly efficient but also robust cell-free system from wheat embryos. We first investigated the source of the instability of existing systems in light of endogenous ribosome-inactivating proteins and found that ribosome inactivation by tritin occurs already during extract preparation and continues during incubation for protein synthesis. Therefore, we prepared our system from extensively washed embryos that are devoid of contamination by endosperm, the source of tritin and possibly other inhibitors. In a batch system, we observed continuous translation for 4 h, and sucrose density gradient analysis showed formation of large polysomes, indicating high protein synthesis activity. When the reaction was performed in a dialysis bag, enabling the continuous supply of substrates together with the continuous removal of small byproducts, translation proceeded for >60 h, yielding 1-4 mg of enzymatically active proteins, and 0.6 mg of a 126-kDa tobacco mosaic virus protein, per milliliter of reaction volume. Our results demonstrate that plants contain endogenous inhibitors of translation and that after their elimination the translational apparatus is very stable. This contrasts with the common belief that cell-free translation systems are inherently unstable, even fragile. Our method is useful for the preparation of large amounts of active protein as well as for the study of protein synthesis itself.