Cell-free translation reconstituted with purified components

@article{Shimizu2001CellfreeTR,
  title={Cell-free translation reconstituted with purified components},
  author={Yoshihiro Shimizu and Akio Inoue and Yukihide Tomari and Tsutomu Suzuki and Takashi Yokogawa and Kazuya Nishikawa and Takuya Ueda},
  journal={Nature Biotechnology},
  year={2001},
  volume={19},
  pages={751-755}
}
We have developed a protein-synthesizing system reconstituted from recombinant tagged protein factors purified to homogeneity. The system was able to produce protein at a rate of about 160 μg/ml/h in a batch mode without the need for any supplementary apparatus. The protein products were easily purified within 1 h using affinity chromatography to remove the tagged protein factors. Moreover, omission of a release factor allowed efficient incorporation of an unnatural amino acid using suppressor… Expand

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References

SHOWING 1-10 OF 30 REFERENCES
Recent advances in producing and selecting functional proteins by using cell-free translation.
TLDR
Progress has been made to efficiently produce proteins in batch or continuous cell-free translation systems and to elucidate the molecular causes of low yield and find possible solutions for this problem. Expand
A continuous cell-free translation system capable of producing polypeptides in high yield.
TLDR
A cell-free translation system has been constructed that uses a continuous flow of the feeding buffer through the reaction mixture and a continuous removal of a polypeptide product, and has proven active for long times. Expand
A highly efficient cell-free protein synthesis system from Escherichia coli.
TLDR
A cell-free coupled transcription/translation system from Escherichia coli with the T7 phage RNA polymerase is modified, and it is found that the optimal concentrations of phosphoenolpyruvate and poly(ethylene glycol) are interdependent; higher concentrations of the former should be used at higher concentrationsof the latter. Expand
Purification and properties of peptidyl-tRNA hydrolase from Escherichia coli.
  • H. Kössel
  • Biology, Medicine
  • Biochimica et biophysica acta
  • 1970
TLDR
The enzyme peptidyl-tRNA hydrolase has been purified from high-speed supernatant or from ribosomes of Escherichia coli yielding a virtually homogeneous product as judged by gel electrophoresis and substrate specificity towards N-acetylaminoacyl-tRNAMet. Expand
Cooperativity of stabilized mRNA and enhanced translation activity in the cell-free system.
TLDR
Detailed analysis of cell-free translation, coupled transcription-translation in static conditions and a continuous flow system based on E. coli S30 extracts found the accurate coupling of transcriptional rate and translational rate was crucial to enhance the efficiency of protein synthesis. Expand
Isolation and point of action of a factor from Escherichia coli required to reconstruct translation.
TLDR
To study the mechanism of translation, the process was reconstructed from purified components and the requirement for a protein called W was demonstrated, which stimulated the synthesis of the hexapeptide, fMet-Ala-Ser-AspNH2-Phe-Thr directed by this RNA. Expand
Effects of release factor 1 on in vitro protein translation and the elaboration of proteins containing unnatural amino acids.
TLDR
An in vitro protein synthesizing system was modified to facilitate the improved, site-specific incorporation of unnatural amino acids into proteins via readthrough of mRNA nonsense (UAG) codons by chemically misacylated suppressor tRNAs, resulting in a substantial increase in mutant protein yields. Expand
DNA-directed in vitro synthesis of beta-galactosidase. Studies with purified factors.
TLDR
The purification from E. coli extracts of three fractions, called Lbeta, Lgamma, and Ldelta, that are needed to restore enzyme synthesis are described, which suggested that other required components were lacking. Expand
A Semicontinuous Prokaryotic Coupled Transcription/Translation System Using a Dialysis Membrane
TLDR
It is proved that the short duration of protein synthesis in a conventional cell‐free protein synthesis system of batch configuration can be attributed both to depletion of energy sources and deactivation of S30 extract by small‐molecule byproducts produced during the protein synthesis. Expand
Rate of elongation of polyphenylalanine in vitro.
Ribosomes purified from Escherichia coli were preincubated with AcPhe-tRNA and poly(U). Then purified components necessary for polypeptide synthesis were added. Incubation of the complete system ledExpand
...
1
2
3
...