Site specific incorporation of keto amino acids into proteins
AssigneeThe Scripps Research Institute
InventorsPeter Schultz, Lei Wang
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Abstract
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 keto amino acids into proteins are provided. Methods for identifying these orthogonal pairs are also provided along with methods of producing proteins with keto amino acids using these orthogonal pairs.
Claims28
A composition comprising an orthogonal aminoacyl-tRNA synthetase (O—RS), wherein the O-RS preferentially aminoacylates an O-tRNA with an efficiency of at least 50% of the efficiency of a polypeptide comprising an amino acid sequence of SEQ ID NO.:
- 18 with a keto amino acid.
A cell comprising a translation system, wherein the translation system comprises:
- an orthogonal-tRNA ( O-tRNA);
- an orthogonal aminoacyl-tRNA synthetase (O—RS);
- and,
- a keto amino acid;
- wherein the O-tRNA recognizes a first selector codon, and the O-RS preferentially aminoacylates the O-tRNA with an efficiency of at least 50% of the efficiency of a polypeptide comprising an amino acid sequence of SEQ ID NO.:
- 18 with the first keto amino acid.
An E. coli cell, comprising:
- an orthogonal tRNA (O-tRNA);
- an orthogonal aminoacyl-tRNA synthetase (O—RS), wherein the O-RS preferentially aminoacylates the O-tRNA with an efficiency of at least 50% of the efficiency of a polypeptide comprising an amino acid sequence of SEQ ID NO.:
- 18 with a keto amino acid;
- the keto amino acid;
- and,
- a nucleic acid that comprises a polynucleotide that encodes a polypeptide of interest, wherein the polynucleotide comprises the selector codon that is recognized by the O-tRNA.
An artificial polypeptide comprising any one of SEQ ID NO. 18-20.
A method of producing a protein in a cell with a keto amino acid at a specified position, the method comprising:
- growing, in an appropriate medium, the cell, where the cell comprises a nucleic acid that comprises at least one selector codon and encodes a protein;
- and,
- providing the keto amino acid;
- wherein the cell further 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 an efficiency of at least 50% of the efficiency of a polypeptide comprising an amino acid sequence of SEQ ID NO.:
- 18 with the keto amino acid;
- and
- incorporating the keto amino acid into the specified position in the protein during translation of the nucleic acid with the at least one selector codon, thereby producing the protein.
Dependencies are inferred from the claim text.