Abstract
In Escherichia coli, conventional amber and ochre stop codons can be separately targeted by engineered amber-suppressing Methanocaldococcus jannaschii tyrosyl-tRNA synthetase-tRNAPyl and ochre-suppressing Methanosarcina maezi pyrrolysyl-tRNA synthetase-tRNAPyl pairs for coding two different noncanonical amino acids in one protein gene. Here, we describe the application of this approach to produce a protein with two distinct chemical functionalites which can be selectively labeled with two fluorescent dyes.
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References
Wang L, Brock A, Herberich B, Schultz PG (2001) Expanding the genetic code of Escherichia coli. Science 292(5516):498–500. https://doi.org/10.1126/science.1060077292/5516/498 [pii]
Wang L, Schultz PG (2004) Expanding the genetic code. Angew Chem 44(1):34–66. https://doi.org/10.1002/anie.200460627
Xie J, Schultz PG (2005) An expanding genetic code. Methods 36(3):227–238. https://doi.org/10.1016/j.ymeth.2005.04.010
Wang L, Xie J, Schultz PG (2006) Expanding the genetic code. Annu Rev Biophys Biomol Struct 35:225–249. https://doi.org/10.1146/annurev.biophys.35.101105.121507
Liu CC, Schultz PG (2010) Adding new chemistries to the genetic code. Annu Rev Biochem 79:413–444. https://doi.org/10.1146/annurev.biochem.052308.105824
Srinivasan G, James CM, Krzycki JA (2002) Pyrrolysine encoded by UAG in Archaea: charging of a UAG-decoding specialized tRNA. Science 296(5572):1459–1462. https://doi.org/10.1126/science.1069588296/5572/1459 [pii]
Blight SK, Larue RC, Mahapatra A, Longstaff DG, Chang E, Zhao G, Kang PT, Green-Church KB, Chan MK, Krzycki JA (2004) Direct charging of tRNA(CUA) with pyrrolysine in vitro and in vivo. Nature 431(7006):333–335. https://doi.org/10.1038/nature02895nature02895 [pii]
Neumann H, Peak-Chew SY, Chin JW (2008) Genetically encoding N(epsilon)-acetyllysine in recombinant proteins. Nat Chem Biol 4(4):232–234. https://doi.org/10.1038/nchembio.73
Wang YS, Fang X, Wallace AL, Wu B, Liu WR (2012) A rationally designed pyrrolysyl-tRNA synthetase mutant with a broad substrate spectrum. J Am Chem Soc 134(6):2950–2953. https://doi.org/10.1021/ja211972x
Wan W, Huang Y, Wang Z, Russell WK, Pai PJ, Russell DH, Liu WR (2010) A facile system for genetic incorporation of two different noncanonical amino acids into one protein in Escherichia coli. Angew Chem Int Ed 49:3211–3214
Wu B, Wang Z, Huang Y, Liu WR (2012) Catalyst-free and site-specific one-pot dual-labeling of a protein directed by two genetically incorporated noncanonical amino acids. Chembiochem 13:1405–1408
Odoi KA, Huang Y, Rezenom YH, Liu WR (2013) Nonsense and sense suppression abilities of original and derivative Methanosarcina mazei pyrrolysyl-tRNA synthetase-tRNAPyl pairs in the Escherichia coli Bl21(DE3) cell strain. PLoS One 8:e57035
Hammill JT, Miyake-Stoner S, Hazen JL, Jackson JC, Mehl RA (2007) Preparation of site-specifically labeled fluorinated proteins for 19F-NMR structural characterization. Nat Protoc 2:2601–2607
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Tharp, J.M., Liu, W.R. (2018). Using Amber and Ochre Nonsense Codons to Code Two Different Noncanonical Amino Acids in One Protein Gene. In: Lemke, E. (eds) Noncanonical Amino Acids. Methods in Molecular Biology, vol 1728. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7574-7_9
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DOI: https://doi.org/10.1007/978-1-4939-7574-7_9
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