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

作者：Ozawa Kiyoshi^*; Loscha Karin V; Kuppan Kekini V; Loh Choy Theng; Dixon Nicholas E; Otting Gottfried

来源：Biochemical and Biophysical Research Communications, 2012, 418(4): 652-656.

DOI：10.1016/j.bbrc.2012.01.069

• Aminoacyl-tRNA-synthetase
• Cell-free protein synthesis
• Escherichia coli
• Suppressor tRNA
• Unnatural amino acids

Using aminoacyl-tRNA synthetase/suppressor tRNA pairs derived from Methanocaldococcus jannaschii, an Escherichia coli cell-free protein production system affords proteins with site-specifically incorporated unnatural amino acids (UAAs) in high yields through the use of optimized amber suppressor tRNA(CUA)(opt) and optimization of reagent concentrations. The efficiency of the cell-free system allows the incorporation of trifluoromethyl-phenylalanine using a polyspecific synthetase evolved previously for p-cyanophenylalanine, and the incorporation of UAAs at two different sites of the same protein without any re-engineering of the E. coli cells used to make the cell-free extract.

• 出版日期2012-2-24