Abstract
The incorporation of noncanonical (unnatural) amino acids into proteins offers researchers the ability to augment the biochemical functionality of proteins for a myriad of applications including bioorthogonal conjugation, biophysical and structural studies, and the enhancement or de novo creation of novel enzymatic activities. The augmentation of a protein throughout its coding sequence by global residue-specific incorporation of unnatural amino acid analogs is an attractive technique for studying both the utility of individual chemistries available through unnatural amino acids and the general effects of unnatural amino acid substitution on protein structure and function. Herein we describe protocols to introduce unnatural amino acids into proteins using the Escherichia coli translation system either in vivo or in vitro. Special attention is paid to obtaining high levels of incorporation while maintaining high yields of protein expression.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Chin JW, Martin AB, King DS, Wang L, Schultz PG (2002) Addition of a photocrosslinking amino acid to the genetic code of Escherichia coli. Proc Natl Acad Sci U S A 99:11020–11024
Chin JW, Santoro SW, Martin AB, King DS, Wang L, Schultz PG (2002) Addition of p-azido-L-phenylalanine to the genetic code of Escherichia coli. J Am Chem Soc 124:9026–9027
Deiters A, Cropp TA, Summerer D, Mukherji M, Schultz PG (2004) Site-specific PEGylation of proteins containing unnatural amino acids. Bioorg Med Chem Lett 14:5743–5745
Lin S, Zhang Z, Xu H, Li L, Chen S, Li J, Hao Z, Chen PR (2011) Site-specific incorporation of photo-cross-linker and bioorthogonal amino acids into enteric bacterial pathogens. J Am Chem Soc 133:20581–20587
Bae JH, Rubini M, Jung G, Wiegand G, Seifert MH, Azim MK, Kim JS, Zumbusch A, Holak TA, Moroder L, Huber R, Budisa N (2003) Expansion of the genetic code enables design of a novel “gold” class of green fluorescent proteins. J Mol Biol 328:1071–1081
Tianero MD, Donia MS, Young TS, Schultz PG, Schmidt EW (2012) Ribosomal route to small-molecule diversity. J Am Chem Soc 134:418–425
Yoo TH, Link AJ, Tirrell DA (2007) Evolution of a fluorinated green fluorescent protein. Proc Natl Acad Sci U S A 104:13887–13890
Johnson JA, Lu YY, Van Deventer JA, Tirrell DA (2010) Residue-specific incorporation of non-canonical amino acids into proteins: recent developments and applications. Curr Opin Chem Biol 14:774–780
Budisa N (2006) Engineering the genetic code. Wiley-VCH, Weinheim
Wong JT (1983) Membership mutation of the genetic code: loss of fitness by tryptophan. Proc Natl Acad Sci U S A 80:6303–6306
Bacher JM, Bull JJ, Ellington AD (2003) Evolution of phage with chemically ambiguous proteomes. BMC Evol Biol 3:24
Bacher JM, Ellington AD (2001) Selection and characterization of Escherichia coli variants capable of growth on an otherwise toxic tryptophan analogue. J Bacteriol 183:5414–5425
Wang L, Brock A, Herberich B, Schultz PG (2001) Expanding the genetic code of Escherichia coli. Science 292:498–500
Hughes RA, Ellington AD (2010) Rational design of an orthogonal tryptophanyl nonsense suppressor tRNA. Nucleic Acids Res 38:6813–6830
Young TS, Schultz PG (2010) Beyond the canonical 20 amino acids: expanding the genetic lexicon. J Biol Chem 285:11039–11044
Taki M, Hohsaka T, Murakami H, Taira K, Sisido M (2001) A non-natural amino acid for efficient incorporation into proteins as a sensitive fluorescent probe. FEBS Lett 507:35–38
Heckler TG, Chang LH, Zama Y, Naka T, Chorghade MS, Hecht SM (1984) T4 RNA ligase mediated preparation of novel “chemically misacylated” tRNAPheS. Biochemistry 23:1468–1473
Kim RG, Choi CY (2001) Expression-independent consumption of substrates in cell-free expression system from Escherichia coli. J Biotechnol 84:27–32
Bacher JM, Ellington AD (2007) Global incorporation of unnatural amino acids in Escherichia coli. Methods Mol Biol 352:23–34
Budisa N, Pal PP, Alefelder S, Birle P, Krywcun T, Rubini M, Wenger W, Bae JH, Steiner T (2004) Probing the role of tryptophans in Aequorea victoria green fluorescent proteins with an expanded genetic code. Biol Chem 385:191–202
Pratt EA, Ho C (1975) Incorporation of fluorotryptophans into proteins of Escherichia coli. Biochemistry 14:3035–3040
Hogue CW, Rasquinha I, Szabo AG, MacManus JP (1992) A new intrinsic fluorescent probe for proteins. Biosynthetic incorporation of 5-hydroxytryptophan into oncomodulin. FEBS Lett 310:269–272
Lark KG (1969) Incorporation of 5-methyltryptophan into the protein of Escherichia coli 15 T- (555-7). J Bacteriol 97:980–982
Kwon I, Tirrell DA (2007) Site-specific incorporation of tryptophan analogues into recombinant proteins in bacterial cells. J Am Chem Soc 129:10431–10437
Schlesinger S (1968) The effect of amino acid analogues on alkaline phosphatase. Formation in Escherichia coli K-12. II. Replacement of tryptophan by azatryptophan and by tryptazan. J Biol Chem 243:3877–3883
Budisa N, Alefelder S, Bae JH, Golbik R, Minks C, Huber R, Moroder L (2001) Proteins with beta-(thienopyrrolyl)alanines as alternative chromophores and pharmaceutically active amino acids. Protein Sci 10:1281–1292
Phillips RS, Cohen LA, Annby U, Wensbo D, Gronowitz S (1995) Enzymatic synthesis of Thia-L-tryptophans. Bioorg Med Chem Lett 5:1133–1134
Hall LE, Hegeman GD, Bosin TR (1974) Incorporation of tryptophan and its benzo(b)thiophene, 1-methylindole, and indene analogs into protein of Escherichia coli. Res Commun Chem Pathol Pharmacol 9:145–153
Acknowledgments
This work was supported by the National Security Science and Engineering Faculty Fellowship (FA9550-10-1-0169), the Welch Foundation (F-1654), and the National Science Foundation (MCB-0943383). R.A.H. is supported by a postdoctoral fellowship from the Cancer Prevention and Research Institute of Texas (Project Nbr: RP101501) and is a postdoctoral fellow of the Applied Research Laboratories at The University of Texas at Austin. The content are solely the responsibility of the authors and do not necessarily represent the official views of the sponsors.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2013 Springer New York
About this protocol
Cite this protocol
Singh-Blom, A., Hughes, R.A., Ellington, A.D. (2013). Residue-Specific Incorporation of Unnatural Amino Acids into Proteins In Vitro and In Vivo. In: Samuelson, J. (eds) Enzyme Engineering. Methods in Molecular Biology, vol 978. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-293-3_7
Download citation
DOI: https://doi.org/10.1007/978-1-62703-293-3_7
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-62703-292-6
Online ISBN: 978-1-62703-293-3
eBook Packages: Springer Protocols