Engineered bacterial tyrosyl-trna synthetase mutants for incorporating unnatural amino acids into proteins
OwnerAbhishek Chatterjee, Katherine T. Grasso +1inventors
InventorsAbhishek Chatterjee, Katherine T. Grasso, Soumya Jyoti Singha Roy
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Abstract
Compositions of bacterial tyrosyl-tRNA synthetase mutants that enable site-specific incorporation of numerous unnatural amino acids into proteins expressed in eukaryotic cells or in engineered ATM E. coli and methods of use are described. Compositions of novel unnatural amino acids that can be incorporated using these mutant bacterial tyrosyl-tRNA synthetase are also described.
Claims21
1-35. (canceled)
A composition comprising a variant E. coli tyrosyl-tRNA synthetase (EcTyr-RS) wherein the variant EcTyr-RS preferentially aminoacacylates an E. coli tyrosyl-RNA (EctRNAtyr) with a tyrosine analog over the naturally-occurring tyrosine amino acid, wherein the variant EcTyr-RS comprises the amino acid sequence of SEQ ID NO:1 or an amino acid sequence with at least about 90% sequence identity with the full-length SEQ ID NO:1, wherein the EcTyr-RS is mutated relative to SEQ ID NO:1 at amino acid residues tyrosine (Y) 37, leucine (L) 71, aspartic acid (D) 182, phenylalanine (F) 183, leucine (L) 186 and aspartic acid (D) 265.
A cell comprising a variant E. coli tyrosyl-tRNA synthetase (EcTyr-RS), wherein the variant EcTyr-RS preferentially aminoacylates an E. coli tyrosyl-tRNA with a tyrosyl analog, and an orthogonal E. coli-tyrosyl tRNA (Ec-tRNATyr) as a pair, wherein the variant EcTyr-RS comprises the amino acid sequence of SEQ ID NO:
- 1 or an amino acid sequence with at least about 90% sequence identity with the full-length SEQ ID NO:1, wherein the EcTyr-RS is mutated relative to SEQ ID NO:
- 1 at amino acid residues tyrosine (Y) 37, leucine (L) 71, aspartic acid (D) 182, phenylalanine (F) 183, leucine (L) 186 and aspartic acid (D) 265.
A method of producing a protein or peptide of interest in an E. coli, eukaryotic or mammalian cell with one, or more, tyrosyl analogs at specified amino acid residue positions in the protein or peptide, the method comprising,
- a. culturing the cell in a culture medium under conditions suitable for growth, wherein the cell comprises a nucleic acid encoding a protein or peptide of interest with one, or more, selector codons incorporated at the one, or more specified positions in the protein or peptide, wherein the cell further comprises a nucleic acid encoding an Ec-tRNATyr that recognizes the selector codon, and
- b. contacting the cell culture medium with one, or more, tyrosyl analogs under conditions suitable for incorporation of the one, or more, tyrosyl analogs into the protein in response to the selector codon,
- thereby producing the protein or peptide of interest with one, or more tyrosyl analogs at specified positions in the protein or peptide.
A method of site-specifically incorporating one, or more, tyrosyl analogs into a protein or peptide of interest in an E. coli, eukaryotic or mammalian cell, the method comprising,
- a. culturing the cell in a culture medium under conditions suitable for growth, wherein the cell comprises a nucleic acid encoding a protein or peptide of interest with one, or more, amber selector codons incorporated at one, or more at specific sites in the protein or peptide, and wherein the cell further comprises a nucleic acid encoding a variant E. coli tyrosyl-tRNA synthetase (EcTyr-RS), wherein the EcTry-RS preferentially aminoacylates an E. coli tyrosyl tRNA (Ec-tRNATry) that recognizes the amber selector codon, wherein the variant EcTyr-RS comprises the amino acid sequence selected from the group consisting of:
- SEQ ID NO:2, SEQ ID NO:
- 3, SEQ ID NO:4 or SEQ ID NO:5;
- and
- b. contacting the cell culture medium with one, or more, tyrosyl analogs under conditions suitable for incorporation of the one, or more, tyrosyl analogs into the protein or peptide at the sites of the selector codon(s),
- thereby producing the protein or peptide of interest with one, or more site-specifically incorporated tyrosyl analogs.
A kit for producing a protein or peptide of interest in a cell, wherein the protein or peptide comprises one, or more tyrosyl analogs, the kit comprising:
- a. a container containing a polynucleotide sequence encoding an Ec-tRNATyr that recognizes an amber selector codon;
- and
- b. a container containing a polynucleotide encoding a variant E. coli tyrosyl tRNA synthetase that preferentially aminoacylates the Ec-tRNATyr with a tryrosyl analog, wherein the EcTry-RS comprises an amino acid sequence selected from the group consisting of:
- SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:
- 4 or SEQ ID NO:5.
Dependencies are inferred from the claim text.