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: 2.28). 02/2012; 418(4):652-6. 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.

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