A New Functional Suppressor tRNA/ Aminoacyl-tRNA Synthetase Pair for the in Vivo Incorporation of Unnatural Amino Acids into Proteins

General methods for selectively incorporating unnatural amino acids into proteins in vivo, directly from the growth media, would greatly expand our ability to manipulate protein structure and function.1 For example, the ability to place fluorophores selectively into proteins in vivo would provide powerful tools for cell biology, or the ability to generate large quantities of proteins with metal binding or keto amino acids might lead to proteins with enhanced physical or catalytic properties. Our approach involves the generation of a suppressor tRNA/aminoacyl-tRNA synthetase (tRNACUA/aaRS) pair that is orthogonal to Escherichia coli endogenous tRNA/synthetase pairs; that is, the orthogonal tRNA is not a substrate for any endogenous synthetases and the orthogonal synthetase does not recognize any endogenous tRNAs.2,3 The specificity of this synthetase is then altered so that it charges the tRNACUA only with a desired unnatural amino acid. One such orthogonal pair for use in E. coli was developed from the tRNA2/GlnRS pair from Saccharomyces cereVisiae.3 The development of additional orthogonal tRNA/aaRS pairs may allow the simultaneous incorporation of multiple unnatural amino acids into proteins. Moreover, different aminoacyl synthetases may be better starting points for generating active sites with particular specificities (e.g., specificity for large hydrophobic vs small hydrophilic amino acids). To this end, we have analyzed biochemical data available for tRNATyr/TyrRS pairs from a variety of organisms. This analysis, together with in vivo complementation assays, has afforded a new orthogonal tRNACUA Tyr /TyrRS pair as well as insights into the development of additional pairs. The identity elements of prokaryotic tRNATyr include a long variable arm in contrast to the short arm of eukaryotic tRNATyr.4 In addition, eukaryotic tRNATyr contains a C1:G72 positive recognition element, whereas prokaryotic tRNATyr has no such consensus base pair.5,6 In vitro studies have also shown that tRNATyr of S. cereVisiae7 and Homo sapiens8 cannot be aminoacylated by bacterial synthetases, nor do their TyrRS aminoacylate bacterial tRNA. To test whether tRNACUA Tyr /TyrRS pairs from these organisms are orthogonal in E. coli, an in vivo complementation assay was used that is based on suppression of an amber stop codon in a nonessential position of the TEM-1 â-lactmase gene encoded in plasmid pBLAM.3 If the newly introduced suppressor tRNACUA is aminoacylated by any endogenous E. coli synthetases, cells will grow in the presence of ampicillin. After expressing these tRNACUA Tyr in E. coli strain DH10B transformed with pBLAM, cells survive at very high concentrations of ampicillin, greater than 1206 μg/mL (interpolated from IC50 curves in Figure 1) for tRNACUA Tyr derived from S. cereVisiae and 234 μg/mL for that from H. sapiens. When S. cereVisiae tRNACUA Gln , which is an orthogonal tRNA, is tested under the same conditions, the cells survive at only 20 μg/mL ampicillin.3 For comparison, E. coli strains bearing pBLAM alone survive up to 9.7 μg/mL ampicillin (in the absence of any suppressor tRNA). Since the recognition of tRNA by synthetase depends on relative concentrations in the cell,9,10 the concentration of tRNACUA Tyr was decreased by expressing its gene under the weaker lac promoter instead of the strong lpp promoter. The IC50’s decreased to 383 and 84 μg/ mL ampicillin for S. cereVisiae and H. sapiens, respectively, but these values are still potentially too high to allow the use of these tRNAs in orthogonal pairs. The change of one single nucleotide in the anticodon (G34 to C34) made the S. cereVisiae and H. sapiens tRNATyr susceptible to acylation by the E. coli synthetases. Most tRNAs have positive and negative elements in the acceptor and anticodon domains to ensure accurate aminoacylation.11,12 Once the anticodon is changed from GUA to CUA, it is possible that noncognate synthetases that recognize tRNAs with similar anticodons have a stronger affinity for tRNACUA Tyr . We hypothesized that the introduction of * To whom correspondence should be addressed. Telephone: (858) 7849300. Fax: (858) 784-9440. E-mail: schultz@scripps.edu. † Present address: Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 02138. (1) Cornish, V. W.; Mendel, D.; Schultz, P. G. Angew. Chem., Int. Ed. Engl. 1995, 34, 621. (2) Liu, D. R.; Magliery, T. J.; Pastrnak, M.; Schultz, P. G. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 10092. (3) Liu, D. R.; Schultz, P. G. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 4780. (4) Himeno, H.; Hasegawa, T.; Ueda, T.; Watanabe, K.; Shimizu, M. Nucleic Acids Res. 1990, 18, 6815. (5) Lee, C. P.; RajBhandary, U. L. Proc. Natl. Acad. Sci. U.S.A. 1991, 88, 11378. (6) Quinn, C. L.; Tao, N.; Schimmel, P. Biochemistry 1995, 34, 12489. (7) Wakasugi, K.; Quinn, C. L.; Tao, N.; Schimmel, P. EMBO J. 1998, 17, 297. (8) Kleeman, T. A.; Wei, D.; Simpson, K. L.; First, E. A. J. Biol. Chem. 1997, 272, 14420. (9) Swanson, R.; Hoben, P.; Sumner-Smith, M.; Uemura, H.; Watson, L.; Soll, D. Science 1988, 242, 1548. (10) Sherman, J. M.; Rogers, M. J.; Soll, D. Nucleic Acids Res. 1992, 20, 1547. (11) Schimmel, P.; Giege, R.; Moras, D.; Yokoyama, S. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 8763. (12) Giege, R.; Sissler, M.; Florentz, C. Nucleic Acids Res. 1998, 26, 5017. Figure 1. In vivo complementation assays to determine the orthogonality of tRNACUA Tyr ’s from various organisms expressed in E. coli. pACXYCUA was used to express tRNACUA Tyr of organism X under the control of the lpp promoter where X ) Y is S. cereVisiae, X ) H is H. sapiens and X ) J is M. jannaschii. pAC-lacXYCUA is similar to pAC-XYCUA except the lpp promoter was changed to lac. pAC-supF was used to express E. coli supF tRNA. pAC is a control plasmid without any tRNA gene. pBLAM was used to express the â-lactamase gene with an amber stop codon at Ala-184. pBLAM-XYRS encodes the indicated TyrRS gene. Numbers following legend text are IC50 values in units of μg/mL ampicillin. 5010 J. Am. Chem. Soc. 2000, 122, 5010-5011