Methods of incorporating amino acid analogs into proteins

摘要

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.

主权项

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 tRNAPhe, 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 tRNAPhe 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 tRNAPhe and the wobble degenerate codon, or the modified tRNAPhe encoded thereby; (2) providing to the translation system a second polynucleotide encoding a modified Aminoacyl tRNA Synthetasel (AARSPhe) with a relaxed substrate specificity where said modified AARSPhe is capable of charging the modified tRNAPhe with an unnatural amino acid in the absence of the natural amino acid or a modified AARSPhe that comprises a mutation at position 415 and is capable of charging the modified tRNAPhe 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 tRNAPhe; 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.