Patent ID: 7713721

Claim:
A method for producing a recombinant orthogonal aminoacyl-tRNA synthetase (O-RS) selective for an unnatural amino acid, the method comprising: (a) mutating a tRNA to improve orthogonality of the tRNA in a host cell, wherein mutating comprises generating a library of tRNAs and selecting and/or screening the library for orthogonality, thereby providing an orthogonal tRNA (O-tRNA); (b) generating a recombinant library of variant homologous RS molecules; (c) selecting or screening the library of variant RSs for members that aminoacylate the orthogonal tRNA (O-tRNA) in the host cell in the presence of an unnatural amino, thereby providing a pool of active RSs; (d) selecting or screening the pool of active RSs to eliminate members of the pool that aminoacylate the O-tRNA with a natural amino acid; and, (e) selecting at least one member of the pool to provide a member with a higher K cat /K m for the unnatural amino acid than for any natural amino acid, thereby providing the at least one recombinant O-RS.