Genetic incorporation of multiple unnatural amino acids into proteins in mammalian cells.

The ability to genetically incorporate unnatural amino acids (UAAs) at specific sites in the proteome of living cells provides a powerful tool to both investigate and engineer protein structure and function. A reassigned nonsense or frameshift codon is used to encode the UAA of interest, and an orthogonal aminoacyl-tRNA synthetase/tRNA (aaRS/ tRNA) pair specific for the UAA delivers the latter cotranslationally into the target protein. This technology has been used to genetically encode a large number of diverse amino acids, including chemically and photochemically reactive amino acids, biophysical probes, metal-ion chelators and redox-active amino acids in E. coli, S. cerevisiae, C. elegans, plant, and mammalian cells. Optimization of the various components of this system in E. coli has resulted in a significant enhancement in suppression efficiency, allowing the incorporation of multiple UAAs in the same polypeptide chain, as well as two different UAAs into a single protein. Unfortunately, suppression systems in mammalian cells are generally less efficient than in E. coli. Furthermore, the incorporation of multiple distinct UAAs into one protein in mammalian cells requires the development of mutually orthogonal aaRS/tRNA pairs capable of suppressing different nonsense/frameshift codons. Here we show that a previously developed enhanced suppression system can be used to incorporate O-methyltyrosine (OMeY, Figure 1b) in good yields at up to three different sites in an enhanced green fluorescent protein (EGFP) in HEK293T cells. We also show that the orthogonal PylRS/tRNAUUA Pyl pair can efficiently suppress the ochre (TAA) nonsense codon in mammalian cells, and in conjunction with the amber (TAG) suppressing EcTyrRS/tRNACUA Tyr pair can be used to incorporate two different UAAs into distinct sites of the same protein. The utility of this technology was demonstrated by generating fulllength anti-HER2 antibody conjugated to auristatin and the fluorophore Alexa Fluor 488 to obtain a defined antibody– drug–fluorophore conjugate. Recently, we reported a mammalian suppression system that offers a significant improvement in the efficiency of UAA incorporation, raising the possibility of its use for the expression of proteins containing multiple UAAs. This system encodes optimized expression cassettes for the aaRS/ tRNA pair and the nonsense mutant of the target gene within one plasmid, which can be used to deliver these genetic elements into mammalian cells either using a baculovirus expression vector, or directly by transient transfection. To evaluate the ability of this system to suppress multiple amber nonsense codons in the same polypeptide, we used pAcBac2 plasmids (Figure 1a) harboring an enhanced green fluorescent protein (EGFP) expression cassette encoding from one to three TAG codons at permissive sites: pAcBac2.tR4OMeYRS/GFP*-1 (Tyr39TAG), pAcBac2.tR4-OMeYRS/ GFP*-2 (Tyr39TAG, Tyr151TAG), and pAcBac2.tR4OMeYRS/GFP*-3 (Ser28TAG, Tyr39TAG, Tyr151TAG). This plasmid also encodes two copies each of the E. coli and Figure 1. Incorporation of one UAA into multiple sites of EGFP expressed in mammalian cells. a) pAcBac2.tR4-OMeYRS/GFP* encodes a CAG-promoter-driven mutant EGFP expression cassette, EcTyrRS gene driven by CMV promoter, and two 2X-tRNACUA Tyr cassettes in two different orientations. b) Structure of O-methyltyrosine (OMeY). c) Expression of the following EGFP mutants in HEK293T cells analyzed by fluorescence microscopy in the presence (+UAA) or absence (-UAA) of 1 mm OMeY. Scale bar=500 mm. d) Expression of the aforementioned EGFP mutants analyzed by SDS-PAGE in the presence (+) or absence ( ) of 1 mm OMeY.