title: Site-specific incorporation of unnatural amino acids into proteins by cell-free protein synthesis.
abstract: Cell-free protein synthesis (CFPS) offers a fast and inexpensive means to incorporate unnatural amino acids (UAAs) site specifically into proteins. This enables engineering of proteins and allows production of protein-based probes for analysis of their interactions with other molecules. Using dialysis Escherichia coli CFPS system in combination with aminoacyl-tRNA synthetase and suppressor tRNA evolved from Methanocaldococcus jannaschii high expression yield of proteins with site specifically incorporated UAAs can be achieved. Typically the target protein can be prepared at concentrations of about 1 mg/mL, which is generally sufficient for subsequent applications.
authors.affiliation: School of Chemistry, University of Wollongong, Wollongong, NSW, Australia.
authors.forename: Kiyoshi
authors.initials: K
authors.lastname: Ozawa
authors.forename: Choy Theng
authors.initials: CT
authors.lastname: Loh
mesh_terms: D000596:Amino Acids / Q000737:chemistry* / Q000378:metabolism*; D000604:Amino Acyl-tRNA Synthetases / Q000378:metabolism; D001386:Azides / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D002457:Cell Extracts; D002474:Cell-Free System; D057930:Click Chemistry; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry; D063747:Methanocaldococcus / Q000201:enzymology / Q000235:genetics; D010649:Phenylalanine / Q000031:analogs & derivatives / Q000378:metabolism; D014176:Protein Biosynthesis; D015202:Protein Engineering / Q000379:methods*; D012343:RNA, Transfer / Q000235:genetics; D013489:Suppression, Genetic / Q000235:genetics
chemical_list: D000596:Amino Acids; D001386:Azides; D002457:Cell Extracts; D005456:Fluorescent Dyes; D010649:Phenylalanine; D012343:RNA, Transfer; D000604:Amino Acyl-tRNA Synthetases