The Experts below are selected from a list of 234 Experts worldwide ranked by ideXlab platform
Hiroaki Imataka - One of the best experts on this subject based on the ideXlab platform.
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Human Cell Extract-derived Cell-free systems for virus synthesis.
Methods in molecular biology (Clifton N.J.), 2013Co-Authors: Tominari Kobayashi, Kodai Machida, Hiroaki ImatakaAbstract:Cell-free synthesis of an infectious virus is an ideal tool for elucidating the mechanism of viral replication and for development of antiviral drugs. In this chapter, the synthesis of Encephalomyocarditis virus (EMCV) from RNA and DNA in a HeLa Cell Extract-derived in vitro protein expression system is described. When a synthetic EMCV RNA with a hammerhead ribozyme sequence at its 5'-end is incubated with a HeLa Cell Extract using a dialysis system, EMCV particles are progressively synthesized. For EMCV synthesis from DNA, a plasmid harboring the full-length cDNA of EMCV with the T7 promoter/terminator unit is incubated in the HeLa Cell Extract supplemented with T7 RNA polymerase.
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Purification and visualization of encephalomyocarditisvirus synthesized by an in vitro protein expression system derived from mammalian Cell Extract
Biotechnology Letters, 2013Co-Authors: Tominari Kobayashi, Kodai Machida, Jun Yukigai, Kosaku Ueda, Mamiko Masutani, Yuri Nishino, Atsuo Miyazawa, Hiroaki ImatakaAbstract:Virus particles are promising vehicles and templates for vaccination, drug delivery and material sciences. Although infectious picornaviruses can be synthesized from genomic or synthetic RNA by Cell-free protein expression systems derived from mammalian Cell Extract, there has been no direct evidence that authentic viral particles are indeed synthesized in the absence of living Cells. We purified encephalomyocarditis virus (EMCV) synthesized by a HeLa Cell Extract-derived, Cell-free protein expression system, and visualized the viral particles by transmission electron-microscopy. The in vitro-synthesized EMCV particles were indistinguishable from the in vivo-synthesized particles. Our results validate the use of the Cell-free technique for the synthesis of EMCV particles.
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an efficient mammalian Cell free translation system supplemented with translation factors
Protein Expression and Purification, 2006Co-Authors: Satoshi Mikami, Mamiko Masutani, Shigeyuki Yokoyama, Nahum Sonenberg, Hiroaki ImatakaAbstract:Abstract Development of an efficient Cell-free translation system from mammalian Cells is an important goal. We examined whether supplementation of HeLa Cell Extracts with any translation initiation factor or translational regulator could enhance protein synthesis. eIF2 (eukaryotic translation initiation factor 2) and eIF2B augmented translation of capped, uncapped and encephalomyocarditis virus-internal ribosome entry site-promoted mRNAs. eIF4E specifically stimulated capped mRNA translation, while p97, a homologue to the C-terminal two-thirds of eIF4G, increased uncapped mRNA translation. When the HeLa Cell Extract was supplemented with a combination of eIF2, eIF2B, and p97, the capacity to synthesize a protein from an uncapped mRNA became comparable to that from the capped counterpart stimulated with a combination of eIF2, eIF2B, and eIF4E. A dialysis method rendered the HeLa Cell Extract capable of synthesizing proteins for 36 h, and the yield was augmented when supplemented with initiation factors. In contrast, the productivity of a rabbit reticulocyte lysate was not enhanced by this method. Collectively, the translation factor-supplemented HeLa Cell Extract should become an important tool for the production of recombinant proteins.
Errol C. Friedberg - One of the best experts on this subject based on the ideXlab platform.
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A yeast whole Cell Extract supports nucleotide excision repair and RNA polymerase II transcription in vitro.
Mutation research, 1996Co-Authors: Zhigang Wang, Errol C. FriedbergAbstract:Abstract Nucleotide excision repair (NER) and RNA polymerase II transcription are Cellular processes that require the transcription/NER factor TFIIH. We have developed a whole Cell Extract from the yeast Saccharomyces cerevisiae that simultaneously supports both NER and RNA polymerase II transcription of independent substrates. NER activity in the yeast whole Cell Extract was readily detected in the absence of further supplementation but was stimulated in the presence of overexpressed Rad2 protein. The repair of N -acetyl-2-aminofluorene (AAF)-damaged DNA was dependent on RAD genes required for NER and deficient repair in rad mutant Extracts was complemented by mixing different mutant Extracts or by purified Rad proteins. Both the NER and transcription activities were stimulated by 5% polyethylene glycol in the whole Cell Extracts. Transcription activity from the template pCYC1G − was not affected by the presence of uracil-containing or AAF-damaged pUC18 DNA, which was expected to result in base excision repair (BER) and NER, respectively. An in vitro condition was defined that supported simultaneous NER and transcription independently in different substrates in the yeast whole Cell Extracts.
Zhigang Wang - One of the best experts on this subject based on the ideXlab platform.
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A yeast whole Cell Extract supports nucleotide excision repair and RNA polymerase II transcription in vitro.
Mutation research, 1996Co-Authors: Zhigang Wang, Errol C. FriedbergAbstract:Abstract Nucleotide excision repair (NER) and RNA polymerase II transcription are Cellular processes that require the transcription/NER factor TFIIH. We have developed a whole Cell Extract from the yeast Saccharomyces cerevisiae that simultaneously supports both NER and RNA polymerase II transcription of independent substrates. NER activity in the yeast whole Cell Extract was readily detected in the absence of further supplementation but was stimulated in the presence of overexpressed Rad2 protein. The repair of N -acetyl-2-aminofluorene (AAF)-damaged DNA was dependent on RAD genes required for NER and deficient repair in rad mutant Extracts was complemented by mixing different mutant Extracts or by purified Rad proteins. Both the NER and transcription activities were stimulated by 5% polyethylene glycol in the whole Cell Extracts. Transcription activity from the template pCYC1G − was not affected by the presence of uracil-containing or AAF-damaged pUC18 DNA, which was expected to result in base excision repair (BER) and NER, respectively. An in vitro condition was defined that supported simultaneous NER and transcription independently in different substrates in the yeast whole Cell Extracts.
Takanori Kigawa - One of the best experts on this subject based on the ideXlab platform.
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Multiple inhibitory factor removal from an Escherichia coli Cell Extract improves Cell-free protein synthesis.
Journal of bioscience and bioengineering, 2009Co-Authors: Eilco Seki, Natsuko Matsuda, Takanori KigawaAbstract:Cell-free protein synthesis systems are generally influenced by the nature of the Cell Extract, which contains various factors on the chromosomal DNA. Some of the Escherichia coli Cell Extract factors are essential, despite their negative effects on protein synthesis, because they are required during the Cell growth and/or Extract preparation stage. In this study, modified E. coli strains were generated by inserting a streptavidin binding peptide (SBP) tag sequence at the 3' termini of the genes encoding polynucleotide phosphorylase (PNPase) and/or Exodeoxyribonuclease V alpha chain (RecD) on the chromosomal DNA. The SBP-tagged target gene products were specifically removed from the Cell Extract prepared from modified E. coli Cells using SBP affinity resin. The linear DNA-directed Cell-free protein synthesis using the treated Extract achieved higher productivity, especially when removing both the PNPase and RecD factors. Using this strategy to remove multiple inhibitory factors in a Cell Extract will be widely applicable to improve Cell-free protein synthesis.
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Preparation of Escherichia coli Cell Extract for highly productive Cell-free protein expression.
Journal of structural and functional genomics, 2004Co-Authors: Takanori Kigawa, Takashi Yabuki, Natsuko Matsuda, Takayoshi Matsuda, Rie Nakajima, Akiko Tanaka, Shigeyuki YokoyamaAbstract:As structural genomics and proteomics research has become popular, the importance of Cell-free protein synthesis systems has been realized for high-throughput expression. Our group has established a high-throughput pipeline for protein sample preparation for structural genomics and proteomics by using Cell-free protein synthesis. Among the many procedures for Cell-free protein synthesis, the preparation of the Cell Extract is a crucial step to establish a highly efficient and reproducible workflow. In this article, we describe a detailed protocol for E. coli Cell Extract preparation for Cell-free protein synthesis, which we have developed and routinely use. The Cell Extract prepared according to this protocol is used for many of our Cell-free synthesis applications, including high-throughput protein expression using PCR-amplified templates and large-scale protein production for structure determinations.
Kei Fujiwara - One of the best experts on this subject based on the ideXlab platform.
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A Relationship between NTP and Cell Extract Concentration for Cell-Free Protein Expression.
Life (Basel Switzerland), 2021Co-Authors: Katsuki Takahashi, Gaku Sato, Nobuhide Doi, Kei FujiwaraAbstract:The Cell-free protein synthesis (CFPS) that synthesizes mRNA and protein from a template DNA has been featured as an important tool to emulate living systems in vitro. However, an obstacle to emulate living Cells by CFPS is the loss of activity in the case of usage of high concentration Cell Extracts. In this study, we found that a high concentration of NTP which inhibits in the case of lower concentration Cell Extract restored the loss of CFPS activity using high concentration Cell Extracts. The NTP restoration was independent of the energy regeneration system used, and NTP derivatives also restored the levels of CFPS using a high concentration Cell Extract. Experiments using dialysis mode of CFPS showed that continuous exchange of small molecule reduced levels of NTP requirement and improved reaction speed of CFPS using the high concentration of Cell Extract. These findings contribute to the development of a method to understand the condition of living Cells by in vitro emulation, and are expected to lead to the achievement of the reconstitution of living Cells from biomolecule mixtures.
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Biochemical Preparation of Cell Extract for Cell-Free Protein Synthesis without Physical Disruption
PloS one, 2016Co-Authors: Kei Fujiwara, Nobuhide DoiAbstract:Cell-free protein synthesis (CFPS) is a powerful tool for the preparation of toxic proteins, directed protein evolution, and bottom-up synthetic biology. The transcription-translation machinery for CFPS is provided by Cell Extracts, which usually contain 20–30 mg/mL of proteins. In general, these Cell Extracts are prepared by physical disruption; however, this requires technical experience and special machinery. Here, we report a method to prepare Cell Extracts for CFPS using a biochemical method, which disrupts Cells through the combination of lysozyme treatment, osmotic shock, and freeze-thaw cycles. The resulting Cell Extracts showed similar features to those obtained by physical disruption, and was able to synthesize active green fluorescent proteins in the presence of appropriate chemicals to a concentration of 20 μM (0.5 mg/mL).
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Condensation of an additive-free Cell Extract to mimic the conditions of live Cells.
PloS one, 2013Co-Authors: Kei Fujiwara, Shin-ichiro M. NomuraAbstract:The Cellular environment differs from that of reconstituted materials mainly because of the presence of highly condensed biomacromolecules. To mimic the environment and conditions in living Cells, we developed a method to prepare additive-free, highly concentrated Cell Extracts. First, we verified the requirement for specific salts and buffers for functional Cell-free translation Extracts. The S30 fraction of Escherichia coli Cell Extracts without additives exhibited sufficient Cell-free protein production. Next, we established a method to accumulate biological components by gradual evaporation by using a vacuum desiccator. Bovine serum albumin, green fluorescent protein, alkaline phosphatase, and a diluted reconstituted protein expression system were successfully condensed in their active forms using this method. The protein concentration of the prepared Cell Extract was elevated to 180 mg/mL, which was expected to contain approximately 260 mg/mL macromolecules, without the loss of Cell-free protein expression activity. Such a condensed Cell Extract may be useful for investigating the differences between Cells and reconstituted materials and may contribute to the development of methods to synthesize Cells from Cell Extracts in the future.