Patent ID: 8715958

Claim:
A method for synthesis of at least one polypeptide containing at least one site specific unnatural amino acid in a cell-free in vitro reaction, the method comprising: synthesizing the polypeptide in a cell-free in vitro translation reaction mix comprising an orthogonal tRNA that base pairs with a nonsense codon, a bacterial cell extract in which ompT is inactive, components of polypeptide and/or mRNA synthesis machinery; a template for transcription of the polypeptide; monomers for synthesis of the polypeptide; and co-factors, enzymes and other reagents necessary for translation; wherein the reaction mix comprises exogenously synthesized orthogonal tRNA synthetase that aminoacylates the orthogonal tRNA with an unnatural amino acid added to the reaction mix at a concentration of at least 30 μg/ml; wherein the polypeptide is synthesized with at least one site specific unnatural amino acid, wherein said reaction mixture produces at least about 50 μg/ml of protein containing at least one site specific unnatural amino acid at a pre-determined site, wherein at least 50% of said protein containing at least one site specific unnatural amino acid is biologically active.