We report a cell-free system for the high-throughput synthesis and screening of gene products. The system, based on the eukaryotic translation apparatus of wheat seeds, has significant advantages over other commonly used cell-free expression systems. To maximize the yield and throughput of the system, we optimized the mRNA UTRs, designed an expression vector for large-scale protein production, and developed a new strategy to construct PCRgenerated DNAs for high-throughput production of many proteins in parallel. The resulting system achieves high-yield expression and can maintain productive translation for 14 days. Additionally, in the integration of a PCR-directed system for template creation, at least 50 genes can be translated in parallel, yielding between 0.1 and 2.3 mg of protein by one person within 2 days. Assessment of correct protein folding by the products of this high-throughput protein-expression system were performed by enzymatic assays of kinases and by NMR spectroscopic analysis. The cell-free system, reported here, bypasses many of the time-consuming cloning steps of conventional expression systems and lends itself to a robotic automation for the high-throughput expression of proteins. With the sequencing of the genomes of various species, attention has turned to the structure, properties, and functional activities of proteins. However, rapid progress in the study of proteins encoded by the newly discovered genes is based on the availability of sufficient amounts of a large number of proteins. Currently, three strategies are being used for protein production: chemical synthesis, in vivo expression, and cell-free protein synthesis. The first two methods have severe limitations: chemical synthesis is not practical for the synthesis of long peptides (1), and in vivo expression can produce only those proteins that do not affect the physiology of the host cell (2). Cell-free translation systems, in contrast, can synthesize large proteins with a speed and accuracy that approach those of in vivo translation (3, 4), and they can express proteins that would otherwise interfere with host cell physiology. However, these systems are reputed to be unstable and thus inefficient (5). One of the most convenient and promising eukaryotic cell-free translation systems is based on wheat embryos that store all of the components of translation in a concentrated dried state, ready for protein synthesis as soon as germination starts. Recently, we found that conventional wheat germ extracts contain the RNA N-glycosidase tritin and other inhibitors of translation such as thionin, ribonucleases, deoxyribonucleases, and proteases. These inhibitors originate from the endosperm (6, 7). Extensive washing of wheat embryos to eliminate endosperm contaminants has resulted in extracts with a high degree of stability and activity. By using mRNA having 5Ј-m 7 GpppG (cap) and a poly(A)-tail (pA) with this extract, the translation reaction proceeds for Ͼ60 h, when it is performed in a dialysis bag with a continuous supply of substrate...

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.

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. ß

Rat liver perchloric acid-soluble protein (L-PSP) is a potent inhibitor of cell-free protein synthesis; however, its mechanism of action is not known. Here we show that the protein is a unique ribonuclease and that this activity is responsible for the inhibition of translation. The addition of perchloric acid-soluble protein to a rabbit reticulocyte cell-free system at a concentration of 6.2 M led to an almost complete inhibition of protein synthesis. The kinetics are unlike those of hemin-controlled inhibitor, a protein that acts at the initiation step. The inhibition appears to be due to an endoribonucleolytic activity of perchloric acid-soluble protein because L-PSP directly affects mRNA template activity and induces disaggregation of the reticulocyte polysomes into 80 S ribosomes, even in the presence of cycloheximide. These effects were observed with authentic as well as recombinant L-PSP. Analysis by thin-layer chromatography of [␣-32 P]UTP-labeled mRNA incubated with the protein showed production of the ribonucleoside 3 -monophosphates Ap, Gp, Up, and Cp, providing direct evidence that the protein is an endoribonuclease. When either 5 -or 3 -32 P-labeled 5 S rRNA was the substrate, L-PSP cleaved phosphodiester bonds only in the singlestranded regions of the molecule.Rat liver perchloric acid-soluble protein (L-PSP) 1 is a 136-amino acid protein that inhibits protein synthesis (1). Oka et al.(1) demonstrated that L-PSP, when added to a rabbit reticulocyte cell-free system, causes inhibition of a biphasic kinetic nature and also leads to the disaggregation of polysomes. This would be similar to the mode of inhibition of translation by the heme-regulated eukaryotic initiation factor 2␣ kinase (2) (however, see our results below). Based on these data, it was suggested that the protein inhibits the initiation step rather than the elongation step (1).A 14-kDa translational inhibitor protein remarkably similar to L-PSP has been characterized in human monocytes and mouse liver (3-5). A homology search revealed that these proteins belong to a new group of small proteins named the YER057c/YJGF family (3), which is of unknown physiological function. The protein sequences of these family members are highly conserved in prokaryotes (including cyanobacteria), fungi, and eukaryotes, suggesting that the proteins may be involved in a basic cellular process. Indeed, mRNA of the translational inhibitor p14.5, the human homologue of L-PSP, becomes significantly up-regulated with the induction of differentiation to macrophages (3), and the synthesis of PSP from rat kidney increases from the 17th fetal day to the fourth postnatal week and then enters a steady-state level (6). In contrast, the expression of PSP from rat kidney in renal tumor cells was down-regulated (6).Recently, Schmiedeknecht et al. (7) have identified the functional promoter of the human p14.5 translational inhibitor. They reported a head-to-head orientation of p14.5 with the gene for the protein subunit hPOP1 of RNase P and with RNase MRP ribonucleoproteins...

G-protein-coupled receptors (GPCRs) are one of the most important drug targets, and anti-GPCR monoclonal antibody (mAb) is an essential tool for functional analysis of GPCRs. However, it is very difficult to develop GPCR-specific mAbs due to difficulties in production of recombinant GPCR antigens, and lack of efficient mAb screening method. Here we describe a novel approach for the production of mAbs against GPCR using two original methods, bilayer-dialysis method and biotinylated liposome-based interaction assay (BiLIA), both of which are developed using wheat cell-free protein synthesis system and liposome technology. Using bilayer-dialysis method, various GPCRs were successfully synthesized with quality and quantity sufficient for immunization. For selection of specific mAb, we designed BiLIA that detects interaction between antibody and membrane protein on liposome. BiLIA prevented denaturation of GPCR, and then preferably selected conformation-sensitive antibodies. Using this approach, we successfully obtained mAbs against DRD1, GHSR, PTGER1 and T1R1. With respect to DRD1 mAb, 36 mouse mAbs and 6 rabbit mAbs were obtained which specifically recognized native DRD1 with high affinity. Among them, half of the mAbs were conformation-sensitive mAb, and two mAbs recognized extracellular loop 2 of DRD1. These results indicated that this approach is useful for GPCR mAb production.