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Cell-free Protein Synthesis System (cell-free + protein_synthesis_system)
Selected AbstractsCell-Free Protein Synthesis System Prepared from Insect Cells by Freeze-ThawingBIOTECHNOLOGY PROGRESS, Issue 6 2006Toru Ezure We established a novel cell-free protein synthesis system derived from Trichoplusia ni (HighFive) insect cells by a simple extraction method. Luciferase and ,-galactosidase were synthesized in this system with active forms. We analyzed and optimized (1) the preparation method of the insect cell extract, (2) the concentration of the reaction components, and (3) the 5,-untranslated region (5,-UTR) of mRNA. The extract was prepared by freeze-thawing insect cells suspended in the extraction buffer. This preparation method was a simple and superior method compared with the conventional method using a Dounce homogenizer. Furthermore, protein synthesis efficiency was improved by the addition of 20% (v/v) glycerol to the extraction buffer. Concentrations of the reaction components were optimized to increase protein synthesis efficiency. Moreover, mRNAs containing 5,-UTRs derived from baculovirus polyhedrin genes showed high protein synthesis activity. Especially, the leader composition of the Ectropis obliqua nucleopolyhedrovirus polyhedrin gene showed the highest enhancement activity among the six 5,-UTRs tested. As a result, in a batch reaction approximately 71 ,g of luciferase was synthesized per milliliter of reaction volume at 25 °C for 6 h. Moreover, this method for the establishment of a cell-free system was applied also to Spodoptera frugiperda 21 (Sf21) insect cells. After optimizing the concentrations of the reaction components and the 5,-UTR of mRNA, approximately 45 ,g/mL of luciferase was synthesized in an Sf21 cell-free system at 25 °C for 3 h. These productivities were sufficient to perform gene expression analyses. Thus, these cell-free systems may be a useful tool for simple synthesis in post-genomic studies as a novel protein production method. [source] Bombyx mori Ras proteins BmRas1, BmRas2 and BmRas3 are neither farnesylated nor palmitoylated but are geranylgeranylatedINSECT MOLECULAR BIOLOGY, Issue 3 2010K. Moriya Abstract The lipid modifications which occur on Bombyx mori Ras proteins BmRas1, BmRas2 and BmRas3 were studied by metabolic labelling in an insect cell-free protein synthesis system and in a baculovirus expression system, using specific inhibitors of protein prenylation and protein palmitoylation. In addition, the subcellular localization of BmRas proteins was examined using EGFP fusion proteins of constitutively active forms of BmRas proteins transiently expressed in Sf9 cells. As a result, it was revealed that the three B. mori Ras proteins BmRas1, BmRas2 and BmRas3 are neither farnesylated nor palmitoylated but are geranylgeranylated for localization to the plasma membrane of insect cells. Thus, the mechanism of membrane binding of insect Ras proteins is quite different from that reported for mammalian Ras proteins. [source] Strategy for comprehensive identification of human N -myristoylated proteins using an insect cell-free protein synthesis systemPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 9 2010Takashi Suzuki Abstract To establish a strategy for the comprehensive identification of human N -myristoylated proteins, the susceptibility of human cDNA clones to protein N -myristoylation was evaluated by metabolic labeling and MS analyses of proteins expressed in an insect cell-free protein synthesis system. One-hundred-and-forty-one cDNA clones with N -terminal Met-Gly motifs were selected as potential candidates from ,2000 Kazusa ORFeome project human cDNA clones, and their susceptibility to protein N -myristoylation was evaluated using fusion proteins, in which the N -terminal ten amino acid residues were fused to an epitope-tagged model protein. As a result, the products of 29 out of 141 cDNA clones were found to be effectively N -myristoylated. The metabolic labeling experiments both in an insect cell-free protein synthesis system and in the transfected COS-1 cells using full-length cDNA revealed that 27 out of 29 proteins were in fact N -myristoylated. Database searches with these 27 cDNA clones revealed that 18 out of 27 proteins are novel N -myristoylated proteins that have not been reported previously to be N -myristoylated, indicating that this strategy is useful for the comprehensive identification of human N -myristoylated proteins from human cDNA resources. [source] Combinatorial, selective and reversible control of gene expression using oligodeoxynucleotides in a cell-free protein synthesis system,BIOTECHNOLOGY & BIOENGINEERING, Issue 2 2009Jung-Won Keum Abstract Herein we describe the methods for selective and reversible regulation of gene expression using antisense oligodeoxynucleotides (ODNs) in a cell-free protein synthesis system programmed with multiple DNAs. Either a complete shut down or controlled level of gene expression was attained through the antisense ODN-mediated regulation of mRNA stability in the reaction mixture. In addition to the primary control of gene expression, we also demonstrate that the inhibition of protein synthesis can be reversed by using an anti-antisense ODN sequence that strips the antisense ODN off the target sequence of mRNA. As a result, sequential additions of the antisense and anti-antisense ODNs enabled the stop-and-go expression of protein molecules. Through the on-demand regulation of gene expression, presented results will provide a versatile platform for the analysis and understanding of the complicated networks of biological components. Biotechnol. Bioeng. 2009;102: 577,582. © 2008 Wiley Periodicals, Inc. [source] A wheat embryo cell-free protein synthesis system not requiring an exogenous supply of GTPBIOTECHNOLOGY PROGRESS, Issue 5 2009Hirohisa Koga Abstract Most in vitro protein synthesis systems require a supply of GTP for the formation of translation initiation complexes, with two GTP molecules per amino acid needed as an energy source for a peptide elongation reaction. In order to optimize protein synthesis reactions in a continuous-flow wheat embryo cell-free system, we have examined the influence of adding GTP and found that the system does not require any supply of GTP. We report here the preparation of a wheat embryo extract from which endogenous GTP was removed by gel filtration, and the influence of adding GTP to the system on protein synthesis reactions. Using Green Fluorescent Protein (GFP) as a reporter, higher levels of production were observed at lower concentrations of GTP, with the optimal level of production obtained with no supply of GTP. A HPLC-based analysis of the extract and the translation mixture containing only ATP as an energy source revealed that GTP was not detectable in the extract, however, 35 ,M of GTP was found in the translation mixture. This result suggests that GTP could be generated from other compounds, such as GDP and GMP, using ATP. A similar experiment with a C-terminally truncated form of human protein tyrosine phosphatase 1B (hPTP1B1-320) gave almost the same result. The wheat embryo cell-free translation system worked most efficiently without exogenous GTP, producing 3.5 mg/mL of translation mixture over a 48-h period at 26°C. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009 [source] Expression of functional Candida antarctica lipase B in a cell-free protein synthesis system derived from Escherichia coliBIOTECHNOLOGY PROGRESS, Issue 2 2009Chang-Gil Park Abstract This article reports the cell-free expression of functional Lipase B from Candida antarctica (CalB) in an Escherichia coli extract. Although most of the cell-free synthesized CalB was insoluble under conventional reaction conditions, the combined use of molecular chaperones led to the soluble expression of CalB. In addition, the functional enzyme was generated by applying the optimal redox potential. When examined using p -nitrophenyl palmitate as a substrate, the specific activity of the cell-free synthesized CalB was higher than that of the reference protein produced in Pichia pastoris. These results highlight the potential of cell-free protein synthesis technology as a powerful platform for the rapid expression, screening and analysis of industrially important enzymes. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009 [source] Cell-Free Protein Synthesis System Prepared from Insect Cells by Freeze-ThawingBIOTECHNOLOGY PROGRESS, Issue 6 2006Toru Ezure We established a novel cell-free protein synthesis system derived from Trichoplusia ni (HighFive) insect cells by a simple extraction method. Luciferase and ,-galactosidase were synthesized in this system with active forms. We analyzed and optimized (1) the preparation method of the insect cell extract, (2) the concentration of the reaction components, and (3) the 5,-untranslated region (5,-UTR) of mRNA. The extract was prepared by freeze-thawing insect cells suspended in the extraction buffer. This preparation method was a simple and superior method compared with the conventional method using a Dounce homogenizer. Furthermore, protein synthesis efficiency was improved by the addition of 20% (v/v) glycerol to the extraction buffer. Concentrations of the reaction components were optimized to increase protein synthesis efficiency. Moreover, mRNAs containing 5,-UTRs derived from baculovirus polyhedrin genes showed high protein synthesis activity. Especially, the leader composition of the Ectropis obliqua nucleopolyhedrovirus polyhedrin gene showed the highest enhancement activity among the six 5,-UTRs tested. As a result, in a batch reaction approximately 71 ,g of luciferase was synthesized per milliliter of reaction volume at 25 °C for 6 h. Moreover, this method for the establishment of a cell-free system was applied also to Spodoptera frugiperda 21 (Sf21) insect cells. After optimizing the concentrations of the reaction components and the 5,-UTR of mRNA, approximately 45 ,g/mL of luciferase was synthesized in an Sf21 cell-free system at 25 °C for 3 h. These productivities were sufficient to perform gene expression analyses. Thus, these cell-free systems may be a useful tool for simple synthesis in post-genomic studies as a novel protein production method. [source] | ||||||||||