Synthesis of Nonnatural mutants of λ-Cro repressor protein that contain an electron-accepting amino acid

The protein biosynthetic system has been extended to incorporate nonnatural amino acids in addition to the 20 naturally occurring ones. Transfer RNAs were chemically aminoacylated with nonnatural amino acids and the aminoacylated tRNAs were added to the cell lysate of E. coli, together with the target mRNA. The positions of the nonnatural amino acids were directed by the 4-base codonlanticodon pairs that are orthogonal to the existing 3-base codonlanticodon pairs. Here, the position-specific incorporation of nonnatural amino acids was applied to incorporate an electron-accepting amino acid, 2anthraquinonylalanine (angAla) into a DNA-binding protein, .-Cro repressor. The position of angAla was directed by a CGGG 4-base colon introduced at several different positions on the mRNA. Among the several mutant proteins, the 64angAla Cro showed a strong binding to the consensus double-stranded DNA.