Methods of incorporating amino acid analogs into proteins

1. A method for incorporating an unnatural amino acid into a target protein at one or more specified position(s), the method comprising:

(1) providing to a translation system a first polynucleotide encoding a modified tRNA Phe , wherein the modified tRNA comprises a modified anticodon sequence that forms Watson-Crick base-pairing with a wobble degenerate codon for a natural amino acid and wherein the modified tRNA Phe interacts with the wobble degenerate codon with an affinity of at least about 1.0 kcal/mole more favorably, at 37° C. than the interaction between unmodified tRNA Phe and the wobble degenerate codon, or the modified tRNA Phe encoded thereby;

(2) providing to the translation system a second polynucleotide encoding a modified Aminoacyl tRNA Synthetasel (AARS Phe ) with a relaxed substrate specificity where said modified AARS Phe is capable of charging the modified tRNA Phe with an unnatural amino acid in the absence of the natural amino acid or a modified AARS Phe that comprises a mutation at position 415 and is capable of charging the modified tRNA Phe with an unnatural amino acid more efficiently than a natural amino acid in the presence of both the natural and unnatural amino acid;

(3) providing to the translation system the unnatural amino acid, wherein the unnatural amino acid is a phenylalanine analog selected from a meta-substituted phenylalanine, or para-substituted phenylalanine, wherein the substitution comprises an aldehyde group, alkenyl group, alkynl group, an azido group, a halogen group, a hydrazide group, a hydrazine group a hydroxyl group, a keto group, a methoxy group, or a methyl group;

(4) providing to the translation system a template polynucleotide encoding the target protein, wherein the codon(s) on the template polynucleotide for the specified position(s) forms Watson-Crick base-pairing with the modified tRNA Phe ; and

(5) allowing translation of the template polynucleotide, thereby incorporating the unnatural amino acid into the target protein at the specified position(s),

wherein steps (1)-(4) are effectuated in any order.

2. The method of claim 1 , wherein the translation system is a cell.

3. The method of claim 1 , wherein the specificity constant (k cat /K M ) for activation of the unnatural amino acid by the modified AARS Phe is at least 5-fold larger than that for the natural amino acid.

4. The method of claim 1 , wherein the modified tRNA Phe is charged by an endogenous AARS Phe at a rate no more than 1% of that of its cognate tRNA Phe .

5. The method of claim 1 , wherein the first polynucleotide and/or the second polynucleotide further comprises either a constitutively active or an inducible promoter sequence that controls the expression of the modified tRNA Phe or AARS Phe , respectively.

6. The method of claim 1 , wherein the translation system is a cell, and the cell is auxotrophic for the natural amino acid encoded at the specified position.

7. The method of claim 1 , wherein the translation system:

(1) lacks endogenous tRNA that forms Watson-Crick base-pairing with the codon(s) at said specified position(s);

(2) is a cell, and the method further comprises disabling one or more genes encoding any endogenous tRNA that forms Watson-Crick base-pairing with the codon(s) at said specified position(s); or

(3) is a cell, and the method further comprises inhibiting one or more endogenous AARS that charges tRNAs that form Watson-Crick base-pairing with the codon(s) at said specified position(s).

8. The method of claim 1 , wherein the cell is a bacterial cell, an E. coli cell, an insect cell, a mammalian cell, a fungal cell, or a yeast cell.

9. The method of claim 1 , wherein the translation system is a cell, and the modified tRNA Phe and/or the modified AARS Phe are derived from an organism different from that of the cell.

10. The method of claim 1 , wherein the phenylalanine analog is incorporated into the position at an efficiency of at least about 50%.

11. The method of claim 2 , wherein the modified tRNA Phe is from an organism different from that of the cell.

12. The method of claim 11 , wherein the modified tRNA Phe is from a yeast, and the cell is an E. coli bacterium.

13. The method of claim 1 , wherein the translation system is a cell, the modified AARS Phe and the tRNA Phe are from the same organism, and the organism is different from the cell.

14. The method of claim 13 , wherein the modified AARS Phe and the tRNA Phe are from a yeast, and the cell is an E. coli bacterium.

15. The method of claim 1 , wherein the wobble degenerate codon is UUU or UUG.