Patent ID: 8778631

Claim:
An in vitro method for introducing non-native amino acids into preselected positions of a polypeptide using a cell-free synthesis system, said method comprising: a) Obtaining a nucleic acid template comprising degenerate sense codons where a first sense codon and a second sense codon correspond to a same native amino acid but differ in their respective nucleotide sequence; b) Generating a cell lysate in a first reaction vessel wherein a native aminoacyl-tRNA synthetase that aminoacylates the native amino acid is depleted by affinity chromatography, immunoaffinity chromatography or immunoprecipitation; c) Adding to the lysate a first non-native aminoacyl-tRNA synthetase that selectively aminoacylates a first isoaccepting sense tRNA with a native amino acid; wherein the first isoaccepting sense tRNA corresponds to the first sense codon of the nucleic acid template; d) Adding a catalytic aminoacylating agent to a second reaction vessel containing a charging reaction mixture including a non-native amino acid and a second isoaccepting sense tRNA corresponding to the second sense codon of the nucleic acid template to generate a tRNA:non-native amino acid charged moiety; e) Combining the cell lysate with: 1) the tRNA:non-native amino acid charged moiety; and, 2) a nucleic acid template comprising the first and second sense codons under conditions appropriate to generate a polypeptide from the template, wherein the polypeptide bears a non-native amino acid in the position corresponding to the second sense codon; and; f) Permitting the reaction to generate the polypeptide bearing non-native amino acids in those positions corresponding to the second sense codons of the nucleic acid template.