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Issue 24, 2011
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Improvement of in vitro-transcribed amber suppressor tRNAs toward higher suppression efficiency in wheat germ extract

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Abstract

In vitro-transcribed, unmodified, and non-aminoacylated amber suppressor tRNAs that are recognized by natural aminoacyl-tRNA synthetase were improved toward higher suppression efficiency in batch-mode cell-free translation in wheat germ extract. The suppression efficiency of the suppressor obtained through four sequence optimization steps (anticodon alteration of natural tRNAs (the first generation); chimerization of the efficient suppressors in the first generation; investigation and optimization of the effective parts in the second generation; combination of the optimized parts in the third generation) and by the terminal tuning was approximately 60%, which was 2.4-fold higher than that of the best suppressor in the first generation. In addition, an eRF1 aptamer further increased the efficiency up to 85%. This highly efficient suppression system also functioned well in a dialysis-based large-scale protein synthesis.

Graphical abstract: Improvement of in vitro-transcribed amber suppressor tRNAs toward higher suppression efficiency in wheat germ extract

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Publication details

The article was received on 09 Aug 2011, accepted on 27 Sep 2011 and first published on 09 Nov 2011


Article type: Paper
DOI: 10.1039/C1OB06351K
Citation: Org. Biomol. Chem., 2011,9, 8495-8503
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    Improvement of in vitro-transcribed amber suppressor tRNAs toward higher suppression efficiency in wheat germ extract

    A. Ogawa, Y. Doi and N. Matsushita, Org. Biomol. Chem., 2011, 9, 8495
    DOI: 10.1039/C1OB06351K

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