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 method for identifying an orthogonal-aminoacyl-tRNA synthetase (O-RS) for use with an O-tRNA that utilizes an unnatural redox amino acid, the method comprising:
subjecting to selection a population of cells of a first species, wherein the cells each comprise:
1) a member of a plurality of aminoacyl-tRNA synthetases (RSs);
2) the orthogonal tRNA (O-tRNA) derived from one or more species; and,
3) a polynucleotide that encodes a selection marker and comprises at least one selector codon recognized by the O-tRNA;
identifying cells that have enhanced suppressor efficiency in the presence of the redox amino acid, as compared to cells lacking the member or comprising a reduced amount of the member of the plurality of RSs, as comprising an active RS that aminoacylates the O-tRNA; and,
selecting the active RS that aminoacylates the O-tRNA with the redox active amino acid, wherein the redox amino acid is an unnatural amino acid selected from the group consisting of: para-substituted tyrosine, ortho-substituted tyrosine, meta substituted tyrosine, para-substituted phenylalanine, ortho-substituted phenylalanine, meta-substituted phenylalanine, 3,4-dihydroxy-L-phenylalanine (DHP), a 3,4,5-trihydroxy-L-phenylalanine, a 3-nitro-tyrosine, and a 3-thiol-tyrosine and selecting the active RS comprises choosing active RS which comprise an amino acid sequence with at least 90% identity to SEQ ID NO: 1; which have a Leu amino acid in a position of the RS corresponding to Tyr32 of SEQ ID NO: 4, have a Ser amino acid residue in a position of the RS corresponding to Ala67 of SEQ ID NO: 4, have an Asn amino acid residue in a position of the RS corresponding to His70 of SEQ ID NO: 4 and have a Gln residue in a position of the RS corresponding to Ala167 of SEQ ID NO: 4; and which aminoacylate the O-tRNA with the redox active amino acid, thereby providing the O-RS for use with the O-tRNA.
2. The method of claim 1 , wherein the selection comprises a positive selection and the selection marker comprises a positive selection marker.
3. The method of claim 1 , wherein the plurality of RSs comprise mutant RSs, RSs derived from one or more species other than the first species or both mutant RS s and RSs derived from a species other than the first species.
4. An orthogonal aminoacyl-tRNA synthetase identified by the method of claim 1 .
5. The method of claim 1 , wherein the plurality of RSs comprises RSs with amino acid substitutions at positions corresponding to position 32, position 67, position 70, position 155, position 158, and position 167 of SEQ ID NO: 4.
6. The method of claim 5 , wherein the redox unnatural amino acid is a meta substituted tyrosine or a meta-substituted phenylalanine.
7. The method of claim 1 , wherein the plurality of RSs comprises RSs with amino acid substitutions at positions corresponding to position 32, position 107, position 158, and position 159 and position 162 of SEQ ID NO: 4.
8. The method of claim 7 , wherein the redox unnatural amino acid is a para-substituted tyrosine or a para-substituted phenylalanine.
9. The method of claim 1 , wherein the selector codon comprises a nonsense or frame shift codon.