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

Ozawa, KiyoshiLoscha, KarinKuppan, Kekini VLoh, ChoyDixon, Nicholas EdwardOtting, Gottfried2015-12-100006-291Xhttp://hdl.handle.net/1885/60845Using 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 tRNACUAopt 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-cyano-phenylalanine, 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.Keywords: amino acid transfer RNA ligase; DnaB helicase; phenylalanine; reagent; article; cell engineering; cell free system; codon; controlled study; enzyme activity; Escherichia coli; in vivo study; Methanocaldococcus jannaschii; nonhuman; priority journal; prote Aminoacyl-tRNA-synthetase; Cell-free protein synthesis; Escherichia coli; Suppressor tRNA; Unnatural amino acidsHigh-yield cell-free protein synthesis for site-specific incorporation of unnatural amino acids at two sites201210.1016/j.bbrc.2012.01.0692016-02-24