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

ArticleinBiochemical and Biophysical Research Communications 418(4):652-6 · February 2012with36 Reads
Impact Factor: 2.30 · DOI: 10.1016/j.bbrc.2012.01.069 · Source: PubMed

    Abstract

    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-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.