Enhanced Mutants of a Bacterial Suppressor Tyrosyl tRNA and Uses Thereof

A method of virus-assisted directed evolution of orthogonal tyrosyl-suppressor tRNA variants of interest with increased biological activity relative to the wild-type suppressor tRNA, wherein the replication of the virus in mammalian cells requires expression of a nonsense mutant of an essential viral protein which is dependent on the activity of the suppressor tRNA variant of interest, the method comprising the steps of:

• a) encoding a library of suppressor tRNA variants in a virus genome;
• b) infecting a population of mammalian host cells with the virus vectors at low multiplicity of infection (MOI);
• c) subsequently introducing into the population of mammalian host cells additional plasmids/genetic elements, wherein the plasmids/genetic elements comprise:
• i) a protein essential for viral replication, wherein a nonsense codon is inserted into the protein sequence rendering viral replication dependent on the activity of the variant suppressor tRNA;
• ii) a cognate Uaa RNA Synthetase (UaaRS);
• and
• iii) genetic components required for viral replication;
• d) substantially simultaneously adding a suitable unnatural amino acid to the culture media;
• e) maintaining the infected/transfected cells in the media under conditions suitable for replication of the virus;
• f) harvesting the cells and isolating virus progeny;
• g) optionally labeling the virus isolated in step f) with a purification handle attached through a photocleavable linker and recovering labeled virus through enrichment followed by release using irradiation at a suitable wavelength;
• h) lysing the recovered virus and amplifying tyrosyl-tRNA variants contained in the lysate whereby orthogonal suppressor tyrosyl-tRNA variants with increased biological activity are recovered.

A method of producing a protein in a mammalian cell with one, or more, tyrosine or phenylalanine amino acid analogs at specified positions in the protein, the method comprising,

• a. culturing the mammalian cell in a culture medium under conditions suitable for growth, wherein the cell comprises a nucleic acid that encodes a protein with one, or more, selector codons,
• wherein the cell further comprises a variant E. coli-derived Tyr-tRNA with increased biological activity that recognizes the selector codon, and its cognate amino acyl-RNA synthetase, and
• b. contacting the cell culture medium with one, or more, tyrosine or phenylalanine amino acid analogs under conditions suitable for incorporation of the one, or more, tyrosine or phenylalanine analogs into the protein in response to the selector codon,
• thereby producing the protein with one, or more tyrosine or phenylalanine analogs incorporated therein.

A method of site-specifically incorporating one, or more, tyrosine or phenylalanine amino acid analogs into a protein or peptide produced in a mammalian cell, the method comprising,

• a. culturing the mammalian cell in a culture medium under conditions suitable for growth, wherein the cell comprises a nucleic acid that encodes a protein or peptide of interest with one, or more, amber, ochre or opal selector codons at specific sites in the protein or peptide,
• wherein the cell further comprises a variant E. coli-derived tRNATyr with increased biological activity that recognizes the selector codon, and further comprises an E. coli Tyr-RNA Synthetase;
• b. contacting the cell culture medium with one, or more, tyrosine or phenylalanine analog residues under conditions suitable for incorporation of the one, or more, tyrosine analog residues into the protein or peptide at the sites of the selector codon(s),
• thereby producing the protein or peptide of interest in a mammalian cell with one, or more site-specifically incorporated tyrosine or phenylalanine analog residues.