Genetic incorporation of unnatural amino acids into proteins in mammalian cells
AssigneeThe Scripps Research Institute
InventorsWenshe Liu, Peter G. Schultz
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Abstract
The invention relates to orthogonal pairs of tRNAs and aminoacyl-tRNA synthetases that can incorporate unnatural amino acids into proteins in mammalian host cells, for example, primate host cells and rodent host cells. The invention provides, for example but not limited to, translation systems that include host cells (e.g., primate or rodent cells), orthogonal aminoacyl-tRNA synthetases derived from eubacterial synthetases, orthogonal tRNAs, and the unnatural amino acid. The invention also relates to methods for producing proteins of interest comprising at least one unnatural amino acid in mammalian host cell systems.
Claims29
A translation system comprising:
- (a) a first unnatural amino acid selected from the group consisting of:
- i) p-methoxyphenylalanine (pMpa);
- ii) p-acetylphenylalanine (pApa);
- iii) p-benzoylphenylalanine (pBpa);
- iv) p-iodophenylalanine (pIpa);
- v) p-azidophenylalanine (pAzpa);
- vi) p-propargyloxyphenylalanine (pPpa);
- vii) α-aminocaprylic acid;
- viii) o-nitrobenzylcysteine (o-NBC);
- ix) 1,5-dansylalanine;
- and
- x) o-nitrobenzylserine (o-NBS);
- (b) a first orthogonal tRNA (O-tRNA);
- (c) a first orthogonal aminoacyl-tRNA synthetase (O-RS) derived from a eubacterial aminoacyl-tRNA synthetase, wherein said O-RS preferentially aminoacylates said O-tRNA with said unnatural amino acid;
- and
- (d) a host cell comprising (a), (b) and (c), said cell selected from a rodent cell and a primate cell.
A method for producing a protein comprising an unnatural amino acid at a selected position, the method comprising:
- (a) providing:
- (i) a first unnatural amino acid selected from the group consisting of:
- A) p-methoxyphenylalanine (pMpa);
- B) p-acetylphenylalanine (pApa);
- C) p-benzoylphenylalanine (pBpa);
- D) p-iodophenylalanine (pIpa);
- E) p-azidophenylalanine (pAzpa);
- F) p-propargyloxyphenylalanine (pPpa);
- G) α-aminocaprylic acid;
- H) o-nitrobenzylcysteine (o-NBC);
- I) 1,5-dansylalanine;
- and
- J) o-nitrobenzylserine (o-NBS);
- (ii) a first orthogonal tRNA (O-tRNA);
- (iii) a first orthogonal aminoacyl-tRNA synthetase (O-RS), wherein said first O-RS preferentially aminoacylates said first O-tRNA with said unnatural amino acid;
- (iv) a nucleic acid encoding said protein, wherein said nucleic acid comprises at least one selector codon that is recognized by said first O-tRNA, and wherein position of selector codon in said nucleic acid corresponds to selected position in said protein;
- and
- (v) a host cell comprising (i), (ii), (iii) and (iv), said host cell selected from a rodent cell and a primate cell;
- and
- (b) incorporating said unnatural amino acid at said selected position in said protein during translation of said protein in response to said selector codon in the presence of said unnatural amino acid, O-tRNA and O-RS, thereby producing said protein comprising said unnatural amino acid at the selected position.
The method of claim 29, wherein exposing the host cell to UV light provides spatial control of protein activity in the host cell.
Dependencies are inferred from the claim text.