Unnatural reactive amino acid genetic code additions
AssigneeThe Scripps Research Institute
InventorsAlexander Deiters, T. Ashton Cropp, Jason W. Chin, J. Christopher Anderson, Peter G. Schultz
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Abstract
This invention provides compositions and methods for producing translational components that expand the number of genetically encoded amino acids in eukaryotic cells. The components include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, orthogonal pairs of tRNAs/synthetases and unnatural amino acids. Proteins and methods of producing proteins with unnatural amino acids in eukaryotic cells are also provided.
Claims9
A method of producing in a eukaryotic cell at least one protein comprising at least one unnatural amino acid, the method comprising:
- growing, in an appropriate medium, a eukaryotic cell that comprises a nucleic acid that comprises at least one selector codon and encodes the protein;
- wherein the medium comprises an unnatural amino acid and the eukaryotic cell comprises:
- an orthogonal tRNA (O-tRNA) that functions in the cell and recognizes the selector codon;
- and,
- an orthogonal aminoacyl tRNA synthetase (O-RS) that preferentially aminoacylates the O-tRNA with the unnatural amino acid, wherein the O-RS comprises the amino acid sequence of any one of SEQ ID NOs:
- 48-53.
A method of producing in a eukaryotic cell at least one protein comprising at least one unnatural amino acid, the method comprising:
- growing, in an appropriate medium, a eukaryotic cell that comprises a nucleic acid that comprises at least one selector codon and encodes the protein;
- wherein the medium comprises the unnatural amino acid and the eukaryotic cell comprises an orthogonal tRNA (O-tRNA) that functions in the cell and recognizes the selector codon and an orthogonal aminoacyl tRNA synthetase (O-RS) that preferentially aminoacylates the O-tRNA with the unnatural amino acid, wherein the O-RS is:
- (i) a p-propargyloxyphenylalanine O-RS that preferentially aminoacylates the O-tRNA with a p-propargyloxyphenylalanine, or
- (ii) a p-azido-L-phenylalanine O-RS that preferentially aminoacylates the O-tRNA with a p-azido-L-phenylalanine, the p-azido-L-phenylalanine O-RS comprising:
- (i) an amino acid sequence set forth in any one of SEQ ID NOs:
- 48-53 and or a conservative variant thereof, wherein the conservative variant is at least 95% identical to any one of SEQ ID NOs:
- 48-53, and comprises two or more amino acids selected from the group consisting of:
- glycine, serine, or alanine at a position corresponding to Tyr37;
- aspartate at a position corresponding to Asn126;
- asparagine at a position corresponding to Asp182;
- alanine, or valine, at a position corresponding to Phe183;
- and, methionine, valine, cysteine, or threonine, at a position corresponding to Leu186;
- or
- (ii) an amino acid sequence that is a conservative variant of SEQ ID NO:
- 2, which conservative variant is at least 98% identical to SEQ ID NO:
- 2 and comprises two or more amino acids selected from the group consisting of:
- glycine, serine, or alanine at a position corresponding to Tyr37;
- aspartate at a position corresponding to Asn126;
- asparagine at a position corresponding to Asp182;
- alanine, or valine, at a position corresponding to Phe183;
- and, methionine, valine, cysteine, or threonine, at a position corresponding to Leu186;
- and,
- incorporating into the protein the unnatural amino acid in the eukaryotic cell, wherein the unnatural amino acid comprises a first reactive group.
Dependencies are inferred from the claim text.