Site-Specific Incorporation of Unnatural Amino Acids into Proteins by Cell-Free Protein Synthesis

2013 ◽  
pp. 189-203 ◽  
Author(s):  
Kiyoshi Ozawa ◽  
Choy Theng Loh
Keyword(s):  
Amino Acids ◽  
Protein Synthesis ◽  
Unnatural Amino Acids ◽  
Site Specific ◽  
Free Protein ◽  
Cell Free Protein Synthesis

scholarly journals High-yield cell-free protein synthesis for site-specific incorporation of unnatural amino acids at two sites

2012 ◽  
Vol 418 (4) ◽  
pp. 652-656 ◽  
Author(s):  
Kiyoshi Ozawa ◽  
Karin V. Loscha ◽  
Kekini V. Kuppan ◽  
Choy Theng Loh ◽  
Nicholas E. Dixon ◽  
...  
Keyword(s):  
Amino Acids ◽  
Protein Synthesis ◽  
Unnatural Amino Acids ◽  
High Yield ◽  
Site Specific ◽  
Free Protein ◽  
Cell Free Protein Synthesis

Translational incorporation of multiple unnatural amino acids in a cell-free protein synthesis system

2014 ◽  
Vol 19 (3) ◽  
pp. 426-432 ◽  
Author(s):  
Su-Jin Oh ◽  
Kyung-Ho Lee ◽  
Ho-Cheol Kim ◽  
Christy Catherine ◽  
Hyungdon Yun ◽  
...  
Keyword(s):  
Amino Acids ◽  
Protein Synthesis ◽  
Unnatural Amino Acids ◽  
Synthesis System ◽  
Free Protein ◽  
A Cell ◽  
Protein Synthesis System ◽  
Cell Free Protein Synthesis

In Situ Deprotection and Incorporation of Unnatural Amino Acids during Cell-Free Protein Synthesis

2013 ◽  
Vol 19 (21) ◽  
pp. 6824-6830 ◽  
Author(s):  
Isaac N. Arthur ◽  
James E. Hennessy ◽  
Dharshana Padmakshan ◽  
Dannon J. Stigers ◽  
Stéphanie Lesturgez ◽  
...  
Keyword(s):  
Amino Acids ◽  
Protein Synthesis ◽  
Unnatural Amino Acids ◽  
Free Protein ◽  
Cell Free Protein Synthesis

scholarly journals Cotranslational incorporation of non-standard amino acids using cell-free protein synthesis

FEBS Letters ◽  
2015 ◽  
Vol 589 (15) ◽  
pp. 1703-1712 ◽  
Author(s):  
Robert B. Quast ◽  
Devid Mrusek ◽  
Christian Hoffmeister ◽  
Andrei Sonnabend ◽  
Stefan Kubick
Keyword(s):  
Amino Acids ◽  
Protein Synthesis ◽  
Free Protein ◽  
Cell Free Protein Synthesis

Einfluß trägergebundener Ribonuclease auf die zellfreie Proteinsynthese im Weizensystem / Influence of Immobilized Ribonuclease on the Cell-free Protein Synthesis in the Wheat System

1975 ◽  
Vol 30 (3-4) ◽  
pp. 227-232 ◽  
Author(s):  
Hartmut Kern
Keyword(s):  
Amino Acids ◽  
Protein Synthesis ◽  
Large Decrease ◽  
Wheat Seedlings ◽  
Free Protein ◽  
Cell Free Protein Synthesis

Abstract In order to increase the efficiency of different mRNAs from wheat seedlings in carrying out cell-free protein synthesis in the wheat system, efforts were made to remove endogenous mRNA. In this direction, we checked the possibility of using immobilized RNAase. Treatment of the cellfree system or its components with this enzyme caused a large decrease in the efficiency of poly Udirected incorporation of labeled amino acids. This effect did not coincide with an equivalent degradation of RNA, as has been shown by analysis of ribosomes and polysomes. The results are discussed in the light of recent findings of some authors

scholarly journals Cell-Free Protein Synthesis Using S30 Extracts from Escherichia coli RFzero Strains for Efficient Incorporation of Non-Natural Amino Acids into Proteins

2019 ◽  
Vol 20 (3) ◽  
pp. 492 ◽  
Author(s):  
Jiro Adachi ◽  
Kazushige Katsura ◽  
Eiko Seki ◽  
Chie Takemoto ◽  
Mikako Shirouzu ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Protein Synthesis ◽  
Trna Synthetase ◽  
Translation Efficiency ◽  
Free System ◽  
Free Protein ◽  
Cell Free System ◽  
Natural Amino Acids ◽  
Cell Free Protein Synthesis

Cell-free protein synthesis is useful for synthesizing difficult targets. The site-specific incorporation of non-natural amino acids into proteins is a powerful protein engineering method. In this study, we optimized the protocol for cell extract preparation from the Escherichia coli strain RFzero-iy, which is engineered to lack release factor 1 (RF-1). The BL21(DE3)-based RFzero-iy strain exhibited quite high cell-free protein productivity, and thus we established the protocols for its cell culture and extract preparation. In the presence of 3-iodo-l-tyrosine (IY), cell-free protein synthesis using the RFzero-iy-based S30 extract translated the UAG codon to IY at various sites with a high translation efficiency of >90%. In the absence of IY, the RFzero-iy-based cell-free system did not translate UAG to any amino acid, leaving UAG unassigned. Actually, UAG was readily reassigned to various non-natural amino acids, by supplementing them with their specific aminoacyl-tRNA synthetase variants (and their specific tRNAs) into the system. The high incorporation rate of our RFzero-iy-based cell-free system enables the incorporation of a variety of non-natural amino acids into multiple sites of proteins. The present strategy to create the RFzero strain is rapid, and thus promising for RF-1 deletions of various E. coli strains genomically engineered for specific requirements.

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