title: High-yield cell-free protein synthesis for site-specific incorporation of unnatural amino acids at two sites.
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.
authors.affiliation: Research School of Chemistry, Australian National University, Canberra, ACT 0200, Australia. ozawa@rsc.anu.edu.au
authors.forename: Kiyoshi
authors.initials: K
authors.lastname: Ozawa
authors.forename: Karin V
authors.initials: KV
authors.lastname: Loscha
authors.forename: Kekini V
authors.initials: KV
authors.lastname: Kuppan
authors.forename: Choy Theng
authors.initials: CT
authors.lastname: Loh
authors.forename: Nicholas E
authors.initials: NE
authors.lastname: Dixon
authors.forename: Gottfried
authors.initials: G
authors.lastname: Otting
mesh_terms: D000409:Alanine / Q000031:analogs & derivatives* / Q000378:metabolism; D000604:Amino Acyl-tRNA Synthetases / Q000235:genetics / Q000378:metabolism*; D002474:Cell-Free System; D018389:Codon, Nonsense; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005818:Genetic Engineering; D017017:Methanococcus / Q000201:enzymology*; D009570:Nitriles / Q000378:metabolism*; D010649:Phenylalanine / Q000031:analogs & derivatives* / Q000378:metabolism; D014176:Protein Biosynthesis; D012343:RNA, Transfer / Q000235:genetics; D013489:Suppression, Genetic
chemical_list: C557397:4-(trifluoromethyl)phenylalanine; D018389:Codon, Nonsense; D009570:Nitriles; C530634:p-cyanophenylalanine; D010649:Phenylalanine; D012343:RNA, Transfer; D000604:Amino Acyl-tRNA Synthetases; D000409:Alanine