Selection of 5′-untranslated sequences that enhance initiation of translation in a cell-free protein synthesis system from wheat embryos

Kamura, Nami; Sawasaki, Tatsuya; Kasahara, Yuko; Takai, Kazuyuki; Endo, Yaeta

doi:10.1016/j.bmcl.2005.09.013

Cited by 49 publications

(27 citation statements)

References 16 publications

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“…DNA encoding full-length PfAMA1 protein was amplified from the genomic DNA of P. falciparum 3D7 and cloned into pEU-E01-GST (a vector with an Nterminal GST tag followed by a tobacco etch virus protease cleavage site) between the XhoI and BamHI sites. These pEU plasmid vectors are the expression vectors designed specifically for the wheat germ cell-free system (16). The inserted nucleotide sequences were confirmed using the ABI PRISM 310 Genetic Analyzer and the BigDye Terminator v1.1 Cycle Sequencing kit (Applied Biosystems, Foster City, CA).…”

Section: Methodsmentioning

confidence: 99%

Wheat Germ Cell-Free System-Based Production of Malaria Proteins for Discovery of Novel Vaccine Candidates

Takeo²,

et al. 2008

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One of the major bottlenecks in malaria research has been the difficulty in recombinant protein expression. Here, we report the application of the wheat germ cell-free system for the successful production of malaria proteins. For proof of principle, the Pfs25, PfCSP, and PfAMA1 proteins were chosen. These genes contain very high A/T sequences and are also difficult to express as recombinant proteins. In our wheat germ cell-free system, native and codon-optimized versions of the Pfs25 genes produced equal amounts of proteins. PfCSP and PfAMA1 genes without any codon optimization were also expressed. The products were soluble, with yields between 50 and 200 g/ml of the translation mixture, indicating that the cell-free system can be used to produce malaria proteins without any prior optimization of their biased codon usage. Biochemical and immunocytochemical analyses of antibodies raised in mice against each protein revealed that every antibody retained its high specificity to the parasite protein in question. The development of parasites in mosquitoes fed patient blood carrying Plasmodium falciparum gametocytes and supplemented with our mouse anti-Pfs25 sera was strongly inhibited, indicating that both Pfs25-3D7/WG and Pfs25-TBV/WG retained their immunogenicity. Lastly, we carried out a parallel expression assay of proteins of blood-stage P. falciparum. The PCR products of 124 P. falciparum genes chosen from the available database were used directly in a small-scale format of transcription and translation reactions. Autoradiogram testing revealed the production of 93 proteins. The application of this new cell-free system-based protocol for the discovery of malaria vaccine candidates will be discussed.Plasmodium falciparum is the protozoan responsible for the widespread return of malaria to tropical countries, particularly in Africa. This reemergence is generally credited to two causes: the development of multidrug-resistant parasites and the development of insecticide-resistant mosquitoes (10). Through decades of work, scientists have learned that vaccination could be a potent curative, but efforts to develop a successful vaccine have not yet succeeded (25). One of the bottlenecks in vaccine development is at the malaria protein production step and is mainly due to the lack of a methodology to enable preparation of quality proteins in an efficient manner. P. falciparum genes have a very high A/T content (average, 76% per gene), and a number of them encode repeated stretches of amino acid sequences (8); these features have been proposed as the major factors limiting P. falciparum protein expression in cell-based systems. Moreover, the presence of glycosylation machinery in eukaryotic cell-based systems can produce inappropriately glycosylated recombinant malaria proteins, resulting in incorrect immune responses (9,21,26). In fact, the three pioneering genome-wide studies on the production of P. falciparum proteins in cell-based systems faced serious problems. For instance, Aguiar et al. (1) were able to obtain exp...

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Section: Methodsmentioning

confidence: 99%

Wheat Germ Cell-Free System-Based Production of Malaria Proteins for Discovery of Novel Vaccine Candidates

Takeo²,

et al. 2008

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“…Figure 1C shows the activities of luciferase translated from five variants (1-5) in wheat germ extract (Madin et al 2000). The variant showing the highest activity is mRNA 2, the translation efficiency of which is z40% compared to that of 59-terminus-mediated translation of mRNA with the most effective enhancer sequence in the 59 UTR (Kamura et al 2005;Ogawa 2009). This variant (2) has the full length of the intergenic region as the IRES, suggesting that the 59 boundary of PSIV IRES is around 6004, which is in accordance with one of previous reports (Kanamori and Nakashima 2001).…”

Section: Optimization Of An Ires-mediated Translation Systemmentioning

confidence: 99%

“…The purified mRNA, which had 251 nucleotides derived from the pE01-Luc in the 39 UTR, was used for translation without polyadenylation and 59-capping (Ogawa 2009). It should be noted that mRNA without polyadenylation and/or 59-capping is efficiently translated in wheat germ extract (Sawasaki et al 2002;Kamura et al 2005;Ogawa 2009). In addition, the ribosome loads on the 59 terminus of mRNA even in the absence of the 59 cap in wheat germ extract (Ogawa 2009).…”

Section: Preparation Of Mrna Templatesmentioning

confidence: 99%

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

Ogawa

2011

RNA

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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 DG 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.

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“…ribosomes, tRNAs, aminoacyl-tRNA synthetases, initiation, elongation and termination factors) [12,13]. The extract is further supplemented with amino acids, energy sources such as ATP, energy generating systems and cofactors for efficient and abundant protein production.…”

Section: Introductionmentioning

confidence: 99%

Molecular and enzymatic characterization of XMRV protease by a cell-free proteolytic analysis

Matsunaga

Sawasaki

Ode

et al. 2012

Journal of Proteomics

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Selection of 5′-untranslated sequences that enhance initiation of translation in a cell-free protein synthesis system from wheat embryos

Cited by 49 publications

References 16 publications

Wheat Germ Cell-Free System-Based Production of Malaria Proteins for Discovery of Novel Vaccine Candidates

Wheat Germ Cell-Free System-Based Production of Malaria Proteins for Discovery of Novel Vaccine Candidates

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

Molecular and enzymatic characterization of XMRV protease by a cell-free proteolytic analysis

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