title: Improvement of in vitro-transcribed amber suppressor tRNAs toward higher suppression efficiency in wheat germ extract.
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.
authors: Senior Research Fellow Center, Ehime University, 3 Bunkyo-cho, Matsuyama, Ehime 790-8577, Japan. a-ogawa@ccr.ehime-u.ac.jp
authors: Atsushi
authors: A
authors: Ogawa
authors: Yasunori
authors: Y
authors: Doi
authors: Nobuto
authors: N
authors: Matsushita
mesh_terms: D002474:Cell-Free System; D016153:Genes, Suppressor; D010936:Plant Extracts / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics; D012343:RNA, Transfer / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology*; D013489:Suppression, Genetic / Q000187:drug effects*; D014908:Triticum / Q000737:chemistry*
chemical_list: D010936:Plant Extracts; D012343:RNA, Transfer