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

Research School of Chemistry, Australian National University, Canberra, ACT 0200, Australia.
Biochemical and Biophysical Research Communications (Impact Factor: 2.3). 02/2012; 418(4):652-6. DOI: 10.1016/j.bbrc.2012.01.069
Source: PubMed


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.

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    • "While hundreds of different non-canonical amino acids have been translationally incorporated into proteins, much of this work has primarily been carried out in vivo (reviewed in (Liu and Schultz, 2010)). Recent advancements in CFPS systems have made in vitro production of proteins containing unnatural amino acids via site-specific incorporation an attractive alternative to in vivo methods (Albayrak and Swartz, 2013a, 2013b; Arthur et al., 2013; Bundy and Swartz, 2010; Goerke and Swartz, 2009; Hong et al., 2014; Loscha et al., 2012; Ozawa et al., 2012). These studies have focused on the site-specific incorporation of unnatural amino acids via the use of engineered translation system components, and the orthogonal suppression of stop-codons (Liu and Schultz, 2010) or "
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