Methods of incorporating amino acid analogs into proteins
The invention provides a method of incorporating nonstandard amino acids into a protein by utilizing a modified aminoacyl-tRNA synthetase to charge the nonstandard amino acid to a modified tRNA, which forms strict Watson-Crick base-pairing with a codon that normally forms wobble base-pairing with natural tRNAs.
1. A polynucleotide encoding a modified tRNA Phe , wherein the modified tRNA Phe 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 has a higher affinity for the wobble degenerate codon than a corresponding wild-type tRNA Phe .
2. The polynucleotide according to claim 1 , wherein the higher affinity interaction of the modified tRNA Phe with the wobble degenerate codon is at least about 1.0 kcal/mole more favorable at 37° C. as compared to the interaction between the wild-type tRNA Phe and the wobble degenerate codon.
3. The polynucleotide according to claim 1 , wherein the wobble degenerate codon is UUU or UUG.
4. The polynucleotide according to claim 1 , wherein the modified tRNA Phe further comprises a mutation at the fourth, extended anticodon site for increasing translation efficiency.
5. A translation system, comprising a polynucleotide of claim 1 , wherein an unnatural amino acid is incorporated into a target protein at a specified position, wherein the unnatural amino acid is a phenylalanine analog.
6. The translation system according to claim 5 , further comprising a second polynucleotide encoding a modified phenylalanine aminoacyl tRNA synthetase (AARS Phe ) with relaxed substrate specificity, wherein the modified AARS Phe 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 acids.
7. The translation system according to claim 5 , comprising more than two different polynucleotides of claim 1 , wherein each of the encoded modified tRNA Phe are each capable of carrying a different unnatural amino acid.
8. The translation system according to claim 6 , wherein the translation system is a cell selected from bacteria, insect, mammalian, fungal, or yeast.
9. The translation system according to claim 8 , wherein the modified tRNA Phe is from an organism different from that of the cell.
10. The translation system according to claim 9 , wherein the modified tRNA Phe is from a yeast, and the cell is an Escherichia coli.
11. The translation system according to claim 8 , wherein the modified AARS Phe and the modified tRNA Phe are from the same organism, wherein the organism is different from that of the cell.
12. The translation system according to claim 9 , wherein the modified AARS Phe and modified tRNA Phe are from a yeast, and the cell is an Escherichia coli.
13. The translation system according to claim 5 , wherein the wobble degenerate codon is UUU or UUG.
14. The translation system according to claim 5 , wherein the higher affinity interaction of the modified tRNA Phe with the wobble degenerate codon is at least about 1.0 kcal/mole more favorable at 37° C. as compared to the interaction between the wild-type tRNA Phe and the wobble degenerate codon.
15. The translation system according to claim 6 , 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.
16. The translation system according to claim 6 , wherein the first polynucleotide, the second polynucleotide or both further comprise either a constitutively active or an inducible promoter sequence that controls expression of the modified tRNA Phe or modified AARS Phe , respectively.
17. The translation system according to claim 8 , wherein the cell is auxotrophic for a natural amino acid encoded at the specified position.
18. The translation system according to claim 5 , wherein the unnatural amino acid comprises a photoactivatable cross-linker or a cycloaddition reactive side group.
19. The translation system according to claim 5 , wherein the phenylalanine analog comprises a meta-substituted phenylalanine or para-substituted phenylalanine, wherein the substitution comprises an alkyl, aryl, acyl, keto, azido, cyano, halo, alkenyl, alkynl, ester, ether, thiol, seleno, sulfonyl, borate, boronate, methoxy, methyl, phospho, phosphono, phosphine, imine, hydrazide, hydrazine, hydroxyl, or aldehyde group.
20. The translation system according to claim 6 , wherein the modified AARS Phe comprises a mutation at position 415.
21. The translation system according to claim 5 , wherein the target protein is an antibody or functional fragment or derivative thereof.
22. A polynucleotide encoding a modified phenylalanine aminoacyl tRNA synthetase (AARS Phe ), wherein the modified AARS Phe comprises a mutation at position 415 and is capable of charging a 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, wherein the modified tRNA Phe comprises a modified anticodon sequence that has higher affinity for a wobble degenerate codon than a corresponding wild-type tRNA Phe .