Patent ID: 7625717

Claim:
A method of producing an orthogonal aminoacyl-tRNA synthetase (O-RS) that preferentially aminoacylates an orthogonal tRNA with an unnatural amino acid in a eukaryotic cell and in the absence of the unnatural amino acid produces less than 30% of a yield of a protein of interest compared to the yield in the presence of the unnatural amino acid, the method comprising: (a) subjecting to positive selection, in the presence of the unnatural amino acid, a population of eukaryotic cells of a first species, wherein the eukaryotic cells each comprise: i) a member of a library of aminoacyl-tRNA synthetases (RSs), ii) an orthogonal tRNA (O-tRNA), iii) a polynucleotide that encodes a positive selection marker that comprises at least one selector codon, which incorporates the unnatural amino acid into the positive selection marker, and iv) a polynucleotide that encodes a negative selection marker that comprises at least one selector codon, which incorporates the unnatural amino acid into the negative selection marker; wherein cells that survive the positive selection comprise an active RS that aminoacylates the orthogonal tRNA (O-tRNA) in the presence of the unnatural amino acid; and (b) subjecting the cells that survive the positive selection to negative selection in the absence of the unnatural amino acid to eliminate active RSs that aminoacylate the O-tRNA with a natural amino acid, thereby providing the O-RS that preferentially aminoacylates the O-tRNA with the unnatural amino acid; and, wherein the polynucleotide that encodes the negative selection marker comprises an ura3 gene and the negative selection can be accomplished on a medium that comprises 5 fluoroorotic acid (5-FOA).