scholarly journals A simple, robust, and low-cost method to produce the PURE cell - free system

2018 ◽  
Author(s):  
Barbora Lavickova ◽  
Sebastian J. Maerkl
Keyword(s):  
Protein Synthesis ◽  
Low Cost ◽  
Translation System ◽  
Free System ◽  
Cell Free System ◽  
Pure System ◽  
Pure Cell ◽  
Synthesis Yield

AbstractWe demonstrate a simple, robust, and low-cost method for producing the PURE cell-free transcription-translation system. Our OnePot PURE system achieved a protein synthesis yield of 156 µg/mL at a cost of 0.09 USD/µL, leading to a 14-fold improvement in cost normalized protein synthesis yield over existing PURE systems. The OnePot method makes the PURE system easy to generate and allows it to be readily optimized and modified.

scholarly journals Cell-Free Protein Synthesis by Diversifying Bacterial Transcription Machinery

BioTech ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 24
Author(s):  
Marina Snapyan ◽  
Sylvain Robin ◽  
Garabet Yeretssian ◽  
Michèle Lecocq ◽  
Frédéric Marc ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Genomic Sequence ◽  
Synthetic Activity ◽  
Translation System ◽  
Free System ◽  
Transcription Terminator ◽  
Cell Free System ◽  
E Coli ◽  
Bacterial Transcription ◽  
A Cell

We have evaluated several approaches to increase protein synthesis in a cell-free coupled bacterial transcription and translation system. A strong pargC promoter, originally isolated from a moderate thermophilic bacterium Geobacillus stearothermophilus, was used to improve the performance of a cell-free system in extracts of Escherichia coli BL21 (DE3). A stimulating effect on protein synthesis was detected with extracts prepared from recombinant cells, in which the E. coli RNA polymerase subunits α, β, β’ and ω are simultaneously coexpressed. Appending a 3′ UTR genomic sequence and a T7 transcription terminator to the protein-coding region also improves the synthetic activity of some genes from linear DNA. The E. coli BL21 (DE3) rna::Tn10 mutant deficient in a periplasmic RNase I was constructed. The mutant cell-free extract increases by up to four-fold the expression of bacterial and human genes mediated from both bacterial pargC and phage pT7 promoters. By contrast, the RNase E deficiency does not affect the cell-free expression of the same genes. The regulatory proteins of the extremophilic bacterium Thermotoga, synthesized in a cell-free system, can provide the binding capacity to target DNA regions. The advantageous characteristics of cell-free systems described open attractive opportunities for high-throughput screening assays.

scholarly journals A Simple, Robust, and Low-Cost Method To Produce the PURE Cell-Free System

2019 ◽  
Vol 8 (2) ◽  
pp. 455-462 ◽  
Author(s):  
Barbora Lavickova ◽  
Sebastian J. Maerkl
Keyword(s):  
Low Cost ◽  
Free System ◽  
Cell Free System ◽  
Pure Cell

Initiation in einem Polyribosomen-abhängigen Protein-synthetisierenden zellfreien System aus Saccharomyces / Initiation in a Polyribosome-Dependent Protein-Synthesizing Cell-Free System from Saccharomyces

1976 ◽  
Vol 31 (3-4) ◽  
pp. 169-173 ◽  
Author(s):  
Bernd Schulz-Harder ◽  
Ernst-Randolf Lochmann
Keyword(s):  
Protein Synthesis ◽  
Free System ◽  
Cell Free System ◽  
Aurintricarboxylic Acid ◽  
Dependent Protein ◽  
A Cell

Abstract A method to prepare polyribosomes from yeasts by using the french-press is described. The highest yield of polyribosomes was derived from late log-phase cells. These polyribosomes, incubated in a cell-free system, were able to reinitiate protein synthesis, which was shown by inhibiting aminoacid incorporation by aurintricarboxylic acid, edeine and sodiumfluoride. We developed the translational system in order to look for the optimal ion-conditions of a DNA-dependent protein-synthesizing system. We found out that at the optimal MgCL2-concentration (6 mᴍ) protein synthesis was strongly inhibited by Mangan ions which are required for transcription in yeast. If protein-synthesis was carried out with 2 mᴍ and 3 mᴍ MgCl2 maximal aminoacid incorporation was observed at 2 mᴍ and 1.5 mᴍ MnCl2.

Enhancement of protein synthesis by an inorganic polyphosphate in an E. coli cell-free system

2006 ◽  
Vol 64 (2) ◽  
pp. 241-249 ◽  
Author(s):  
Hiromichi Itoh ◽  
Yumi Kawazoe ◽  
Toshikazu Shiba
Keyword(s):  
Protein Synthesis ◽  
Coli Cell ◽  
Inorganic Polyphosphate ◽  
Free System ◽  
Cell Free System ◽  
E Coli

scholarly journals Buffer preparation for OnePot PURE cell-free system v1 (protocols.io.728hqhw)

protocols.io ◽  
2019 ◽  
Author(s):  
Konstantinos Ragios
Keyword(s):  
Free System ◽  
Cell Free System ◽  
Pure Cell

scholarly journals Temperature dependency of protein synthesis by cell-free system constructed with polysomes from rice radicle.

1987 ◽  
Vol 56 (1) ◽  
pp. 44-50 ◽  
Author(s):  
Junichi TAKANASHI ◽  
Sachio MARUYAMA ◽  
Nobuyuki KABAKI ◽  
Koichi TAJIMA
Keyword(s):  
Protein Synthesis ◽  
Temperature Dependency ◽  
Free System ◽  
Cell Free System

scholarly journals The Capacity of Polyadenylated RNA from Myogenic Cells Treated with Actinomycin D to Direct Protein Synthesis in a Cell-Free System

1978 ◽  
Vol 88 (2) ◽  
pp. 403-410 ◽  
Author(s):  
Gania KESSLER-ICEKSON ◽  
Robert H. SINGER ◽  
David YAFFE
Keyword(s):  
Protein Synthesis ◽  
Actinomycin D ◽  
Myogenic Cells ◽  
Free System ◽  
Cell Free System ◽  
Polyadenylated Rna ◽  
A Cell

Studies on the Incorporation of Amino Acids by a Cell-Free System Obtained from Beef Retina

1972 ◽  
Vol 50 (5) ◽  
pp. 581-587 ◽  
Author(s):  
Y. Matuk
Keyword(s):  
Amino Acids ◽  
Protein Synthesis ◽  
Incubation Medium ◽  
Optimum Concentration ◽  
Free System ◽  
Cell Free System ◽  
A Cell

The incorporation of 14C-leucine into proteins by a cell-free system from beef retina was studied. It was found that the optimum concentration of ATP depended on the concentration of ribosomes in the incubation medium. Very little incorporation of 14C-leucine was observed in the absence of K+. The optimum concentration of phosphocreatine required for incorporation of radioactive leucine depended on the concentration of Mg2+ in the incubation medium, and the optimum concentration of K+ appears to be independent of the concentrations of Mg2+ and phosphocreatine used.Retinol and retinal had no effect, but ethanol markedly inhibited protein synthesis at concentrations higher than 2%.Puromycin (10−4 M) inhibited incorporation of 14C-leucine by about 80%. The degree of inhibition by cycloheximide depended on the concentration of pH 5 fraction in the incubation medium.

scholarly journals Protein synthesis in a cell-free system coded by poly(A)-mRNA isolated from bovine retina

1981 ◽  
Vol 31 ◽  
pp. 98
Author(s):  
Che-Hui Kuo ◽  
Naomasa Miki
Keyword(s):  
Protein Synthesis ◽  
Free System ◽  
Cell Free System ◽  
Bovine Retina ◽  
A Cell

Initiation of protein synthesis in a cell-free system prepared from rat hepatocytes

1989 ◽  
Vol 256 (1) ◽  
pp. C28-C34 ◽  
Author(s):  
S. R. Kimball ◽  
W. V. Everson ◽  
K. E. Flaim ◽  
L. S. Jefferson
Keyword(s):  
Protein Synthesis ◽  
Amino Acid ◽  
Rat Hepatocytes ◽  
Initiation Factor ◽  
Nucleotide Exchange ◽  
Isolated Rat Hepatocytes ◽  
Intact Cells ◽  
Free System ◽  
Cell Free System ◽  
A Cell

A cell-free system, which maintained a linear rate of protein synthesis for up to 20 min of incubation, was prepared from isolated rat hepatocytes. The rate of protein synthesis in the cell-free system was approximately 20% of the rate obtained in isolated hepatocytes or perfused liver. More than 70% of total protein synthesis in the cell-free system was due to reinitiation, as indicated by addition of inhibitors of initiation, i.e., edeine or polyvinyl sulfate. The rate of protein synthesis and formation of 43S initiation complexes in the cell-free system were reduced to 60 and 30% of the control values, respectively, after incubation of hepatocytes in medium deprived of an essential amino acid. Therefore, the cell-free system maintained the defect in initiation induced in the intact cells by amino acid deprivation. The defect in initiation was corrected by addition of either rat liver eukaryotic initiation factor 2 or the guanine nucleotide exchange factor (GEF) to the cell-free system. A role for GEF in the defect in initiation was further implicated by experiments that showed that the activity of the factor was decreased in extracts from livers perfused with medium deficient in amino acids. The cell-free system should provide a valuable tool for investigation of mechanisms involved in the regulation of initiation of protein synthesis.

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