Site-specific incorporation of redox active amino acids into proteins
Compositions and methods of producing components of protein biosynthetic machinery that include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases, which incorporate redox active amino acids into proteins are provided. Methods for identifying these orthogonal pairs are also provided along with methods of producing proteins with redox active amino acids using these orthogonal pairs.
1. A composition comprising an orthogonal aminoacyl-tRNA synthetase (O-RS) wherein the ORS preferentially aminoacylates an O-tRNA with a meta-substituted tyrosine unnatural amino acid,
wherein the O-RS comprises an amino acid sequence with at least 90% identity to SEQ ID NO: 1,
wherein the O-RS further comprises a Leu amino acid in a position of the O-RS corresponding to Tyr32 of SEQ ID NO: 4, a Ser amino acid residue in a position of the O-RS corresponding to Ala67 of SEQ ID NO: 4, an Asn amino acid residue in a position of the O-RS corresponding to His70 of SEQ ID NO: 4 and a Gln residue in a position of the O-RS corresponding to Ala167 of SEQ ID NO: 4; and SEQ ID NO: 4 is the wild type sequence.
2. The composition of claim 1 , wherein the meta-substituted tyrosine is selected from the group consisting of: a 3,4-dihydroxy-L-phenylalanine (DHP) a 3,4,5-trihydroxy-L-phenylalanine, a 3-nitro-tyrosine, and a 3-thiol-tyrosine.
3. The composition of claim 1 , wherein the O-RS is derived from a Methanococcus jannaschii.
4. The composition of claim 1 , comprising a cell.
5. The composition of claim 1 , comprising a translation system.