A R T I C L E S Figure 3. MmtDNACPyUlA is orthogonal in yeast, but MbtDNAPyl CUA directs the incorporation of alanine and is not orthogonal in yeast. (A) Constructs used to compare orthogonality of tRNACPyUlA in yeast. (B) Analysis of amber suppression by expression of hSOD33TAG-His6 and detection by anti-His6 western blot. Yeast cells containing the hSOD expression plasmid, MbPylRS and the dicistronic SctDNAArg UCU -tDNAPyl CUA construct were grown in the presence or absence of 1 (5 mM). (C) ESI-MS shows that alanine is incorporated into hSOD33TAG in cells producing amber suppressor MbtDNACPyUlA from construct 7 (Found 16553 ( 1.5 Da, expected 16553 Da), conﬁrming that MbtDNACPyUlA is a substrate for yeast alanyl-tRNA synthetases. phenotypes even in the absence of added amino acid 1: construct 5 is blue on X-Gal in the presence and absence of 1, and construct 7 is blue in the presence and absence of 1 and grows on media lacking histidine and containing 3AT in the presence and absence of 1. These experiments revealed that MbtRNAPCyUlA is not orthogonal in yeast. To identify the molecular basis of the non-orthogonality of MbtDNACPyUlA we examined the sequence of MbtRNACPyUlA for nucleotides that match the positive identity elements within yeast tRNAs that are speciﬁcally recognized by yeast synthetases.20 We realized that MbtRNAPCyUlA contains an unusual G3 · U70 base pair, which is a positive identity element by which yeast alanyl- tRNA synthetase recognizes SctRNAAla.21 This suggested that MbtRNAPCyUlA may be aminoacylated by alanyl-tRNA synthetase in yeast. To test this hypothesis we expressed human superoxide dismutase (hSOD) bearing an amber codon at position 33 (from pC1 hSOD33TAG-His6 in the MJY125-derived strain SCY422). Expression of hSOD was dependent on the presence of SctDNAUACrgU-MbtDNACPyUlA, but did not decrease substantially in the absence of 1 (Figure 3B), further conﬁrming that the SctDNAAUrCgU-MbtDNAPCyUlA cassette confers efﬁcient amber sup- pression, which is not dependent on MbPylRS. ESI-MS spectra of hSOD puriﬁed from expressions using MbPylRS/MbtRNAPCyUlA in the absence of unnatural amino acid were consistent with the incorporation of alanine in response to the amber codon in (20) Giege´, R.; Sissler, M.; Florentz, C. Nucleic Acids Res. 1998, 26, 5017. (21) (a) Hou, Y. M.; Schimmel, P. Nature 1988, 333, 140. (b) McClain, W. H.; Foss, K. Science 1988, 240, 793. (22) Summerer, D.; Chen, S.; Wu, N.; Deiters, A.; Chin, J. W.; Schultz, P. G. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 9785. 14822 J. AM. CHEM. SOC. 9 VOL. 132, NO. 42, 2010 Hancock et al. hSOD33TAG (Figure 3C), conﬁrming our hypothesis on the molecular basis of MbtDNACPyUlA non-orthogonality. To create an MbtDNAPCyUlA construct that is orthogonal in yeast we converted the G3 · U70 base pair in MbtRNAPCyUlA to A3-U70. This changes MbtRNAPCyUlA to MmtRNAPCyUlA (Figure 3A construct 8). Yeast containing MbPylRS/SctDNAAUrCgU-MmtDNAPCyUlA pro- duced full-length hSOD-His6 from pC1SOD33TAG only in the presence of 1 (Figure 3B). These experiments establish that functional MmtRNACPyUlA is produced from the dicistronic construct and is orthogonal in yeast. To begin to demonstrate the range of amino acids that can be incorporated in yeast using our approach, we incorporated the important post-translational modiﬁcation Nε-acetyl-L-lysine (2) and its analog Nε-triﬂuoroacetyl-L-lysine (3), a photocaged lysine derivative Nε-[(1-(6-nitrobenzo[d][1,3]dioxol-5-yl)ethoxy)- carbonyl]-L-lysine (4), and photo-cross-linker Nε-[(2-(3-methyl- 3H-diazirin-3-yl)ethoxy)carbonyl]-L-lysine (5) into hSOD-His6 produced in S. cereVisiae (Figure 4A) using MbPylRS and variants of MbPylRS we have previously evolved in E. coli.1-3 While we have not speciﬁcally evolved a synthetase for Nε- triﬂuoroacetyl-L-lysine, we have found that AcKRS2,1 previ- ously evolved for incorporating Nε-acetyl-L-lysine, efﬁciently incorporates this amino acid. We demonstrated the incorporation of each amino acid by western blot (Figure 4A). We carried out large-scale expression and puriﬁcation of hSOD in the presence of 1, 2, and 3 (Figure 4B), which unlike 4 and 5 are not photosensitive and are available in gram quantities, to further conﬁrm the site and identity of amino acid incorporation by ESI-MS and MS/MS sequencing (Figure 4C-H). We have demonstrated the speciﬁc incorporation of an amino acid into SOD in the presence of 4 and 5. In addition we have reported MS and MS/MS data for the incorporation of amino acids 4 and 5 into proteins in other organisms.2,23 However, we have not yet obtained MS data directly in yeast and cannot rule out the possibility that an aspect of yeast metabolismsthat is not conserved in either other eukaryotes or bacteriasleads to the selective post-translational modiﬁcation of these amino acids in ViVo. Puriﬁed hSOD yields were 30-100 µ g/L of yeast culture which is similar to the 50 µ g/L yield reported for incorporating p-acetyl-L-phenylalanine into hSOD using the EcTyrRS/tRNACTyUrA pair in yeast.8 Conclusions In summary, we have solved the key challenges of producing a functional and orthogonal tRNACPyUlA in yeast. We have discovered an MbPylRS/tRNACPyUlA pair that is orthogonal in yeast, and described a simple system through which variant MbPylRS/tRNACPyUlA pairs created in E. coli can be transplanted to expand the genetic code of yeast for a wide range of unnatural amino acids. Using our approach we have incorporated the alkyne-containing amino acid Nε-[(2-propynyloxy)carbonyl]-L- lysine (1), an important post-translationally modiﬁed amino acid Nε-acetyl-L-lysine (2), and an analog of Nε-acetyl-L-lysine, triﬂuoroacetyl-L-lysine (3), a photocaged lysine derivative Nε- [(1-(6-nitrobenzo[d][1,3]dioxol-5-yl)ethoxy)carbonyl]-L-lysine (4), and a photo-cross-linker Nε-[(2-(3-methyl-3H-diazirin-3- yl)ethoxy)carbonyl]-L-lysine (5) into proteins in yeast. Amino acid 1 may be used for bio-orthogonal [3 + 2] cycloadditions (23) Chou, C.; Uprety, R.; Davis, L.; Chin, J. W.; Deiters, A. Submitted 2010.