Stable cell lines for site-specific incorporation of unnatural amino acids

A eukaryotic cell line capable of expressing a target protein containing at least one unnatural amino acid from a gene containing a premature stop codon at a position corresponding to the position for incorporation of the unnatural amino acid, the cell line comprising a genome having stably integrated therein (i) a nucleic acid sequence encoding a prokaryotic tryptophanyl-tRNA synthetase mutein capable of charging a tRNA with an unnatural amino acid and (ii) a nucleic acid sequence encoding a prokaryotic suppressor tryptophanyl-tRNA capable of being charged with the unnatural amino acid.

A prokaryotic leucyl tRNA synthetase mutein capable of charging a tRNA with an unnatural amino acid for incorporation into a protein, the tRNA synthetase mutein comprising the amino acid sequence of SEQ ID NO:

• 1 and (i) at least one substitution (e.g., a substitution with a hydrophobic amino acid) at a position corresponding to His537, (ii) at least one amino acid substitution selected from E20V, E20M, L41V, L41A, Y499H, Y499A, Y527I, Y527V, Y527G, and any combination thereof, (iii) at least one amino acid substitution selected from E20K and L41S and any combination thereof and at least one amino acid substitution selected from M40I, T252A, Y499I, and Y527A, and any combination thereof, or (iv) a combination of two or more of (i), (ii) and (iii).