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US 6,586,207 B2Granted
Overexpression of aminoacyl-tRNA synthetases for efficient production of engineered proteins containing amino acid analogues
AssigneeCalifornia Institute of Technology
InventorsDavid A. Tirrell, Kristi Lynn Kiick
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Abstract
Methods for producing modified polypeptides containing amino acid analogues are disclosed. The invention further provides purified dihydrofolate reductase polypeptides, produced by the methods of the invention, in which the methionine residues have been replaced with homoallyglycine, homoproparglycine, norvaline, norleucine, cis-crotylglycine, trans-crotylglycine, 2-aminoheptanoic acid, 2-butynylglycine and allylglycine.
Claims14
A method for producing a modified polypeptide, the polypeptide being modified by replacing a selected amino acid with a desired amino acid analogue, which method comprises:
- a) transforming a host cell with:
- i) a vector having a polynucleotide sequence encoding an aminoacyl-tRNA synthetase for the selected amino acid;
- and
- ii) a vector having a polynucleotide sequence encoding a polypeptide molecule or interest so as to produce a host vector system;
- wherein the vectors of (i) and (ii) may be the same or different
- b) growing the host-vector system in a medium which comprises the selected amino acid so that the host vector system overexpresses the aminoacyl-tRNA synthetase;
- c) replacing the medium with a medium which lacks the selected amino acid and has the desired amino acid analogue;
- d) growing the host vector system in the medium which lacks the selected amino acid and has the desired amino acid analogue under conditions so that the host vector system overexpresses the polypeptide molecule of interest and the selected amino acid is replaced with the desired amino acid analogue thereby producing the modified polypeptide.
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Dependencies are inferred from the claim text.