Global Incorporation of Unnatural Amino Acids in Escherichia coli

  • Jamie M. Bacher
  • Andrew D. Ellington
Part of the Methods in Molecular Biology™ book series (MIMB, volume 352)

Abstract

The incorporation of amino acid analogs is becoming increasingly useful. Site-specific incorporation of unnatural amino acids allows the application of chemical biology to protein-specific investigations and applications. However, the global incorporation of unnatural amino acids allows for tests of proteomic and genetic code hypotheses. For example, the adaptation of organisms to unnatural amino acids may lead to new genetic codes. To understand and quantify changes from such perturbations, an understanding is required of the microbiological and proteomic responses to the incorporation of unnatural amino acids. Here we describe protocols to characterize the effects of such proteome-wide perturbations.

Key Words

Unnatural amino acids genetic code ambiguity amino acid misincorporation amino acylation errors amino acid analogs genetic code evolution 

References

  1. 1.
    Wang, L., Zhang, Z., Brock, A., and Schultz, P. G. (2003) Addition of the keto functional group to the genetic code of Escherichia coli. Proc. Natl. Acad. Sci. USA 100, 56–61.PubMedCrossRefGoogle Scholar
  2. 2.
    Budisa, N., Steipe, B., Demange, P., Eckerskorn, C., Kellermann, J., and Huber, R. (1995) High-level biosynthetic substitution of methionine in proteins by its analogs 2-aminohexanoic acid, selenomethionine, telluromethionine and ethionine in Escherichia coli. Eur. J. Biochem. 230, 788–796.PubMedCrossRefGoogle Scholar
  3. 3.
    Bacher, J. M., Hughes, R. A., Tze-Fei Wong, J., and Ellington, A. D. (2004) Evolving new genetic codes. Trends Ecol. Evol. 19, 69–75.Google Scholar
  4. 4.
    Bacher, J. M., Bull, J. J., and Ellington, A. D. (2003) Evolution of phage with chemically ambiguous proteomes. BMC Evolutionary Biol. 3, 24.CrossRefGoogle Scholar
  5. 5.
    Bacher, J. M. and Ellington, A. D. (2001) Selection and characterization of escherichia coli variants capable of growth on an otherwise toxic tryptophan analogue. J. Bacteriol. 183, 5414–5425.PubMedCrossRefGoogle Scholar
  6. 6.
    Wong, J. T. (1983) Membership mutation of the genetic code: loss of fitness by tryptophan. Proc. Natl. Acad. Sci. USA 80, 6303–6306.PubMedCrossRefGoogle Scholar
  7. 7.
    Bronskill, P. M. and Wong, J. T. (1988) Suppression of fluorescence of tryptophan residues in proteins by replacement with 4-fluorotryptophan. Biochem. J. 249, 305–308.PubMedGoogle Scholar
  8. 8.
    Parsons, J. F., Xiao, G., Gilliland, G. L., and Armstrong, R. N. (1998) Enzymes harboring unnatural amino acids: mechanistic and structural analysis of the enhanced catalytic activity of a glutathione transferase containing 5-fluorotryptophan. Biochemistry 37, 6286–6294.PubMedCrossRefGoogle Scholar
  9. 9.
    Pratt, E. A. and Ho, C. (1975) Incorporation of fluorotryptophans into proteins of escherichia coli. Biochemistry 14, 3035–3040.PubMedCrossRefGoogle Scholar
  10. 10.
    Zhang, Q. S., Shen, L., Wang, E. D., and Wang, Y. L. (1999) Biosynthesis and characterization of 4-fluorotryptophan-labeled Escherichia coli arginyl-tRNA synthetase. J. Protein Chem. 18, 187–192.PubMedCrossRefGoogle Scholar
  11. 11.
    Browne, D. R., Kenyon, G. L., and Hegeman, G. D. (1970) Incorporation of monoflurotryptophans into protein during the growth of Escherichia coli. Biochem. Biophys. Res. Commun. 39, 13–19.PubMedCrossRefGoogle Scholar
  12. 12.
    Guan, K. L. and Dixon, J. E. (1991) Eukaryotic proteins expressed in Escherichia coli: an improved thrombin cleavage and purification procedure of fusion proteins with glutathione S-transferase. Anal. Biochem. 192, 262–267.PubMedCrossRefGoogle Scholar
  13. 13.
    Xu, Z. J., Love, M. L., Ma, L. Y., et al. (1989) Tryptophanyl-tRNA synthetase from Bacillus subtilis. Characterization and role of hydrophobicity in substrate recognition. J. Biol. Chem. 264, 4304–4311.PubMedGoogle Scholar
  14. 14.
    Hendrickson, T. L., Nomanbhoy, T. K., de Crécy-Lagard, V., et al. (2002) Mutational separation of two pathways for editing by a class I tRNA synthetase. Mol. Cell. 9, 353–362.PubMedCrossRefGoogle Scholar
  15. 15.
    Bacher, J. M., de Crécy-Lagard, V., and Schimmel, P. (2005) Inhibited cell growth and protein functional changes from an editing-defective tRNA synthetase. Proc. Natl. Acad. Sci. USA 102, 1697–1701.PubMedCrossRefGoogle Scholar
  16. 16.
    King, P. V. and Blakesly, R. W. (1986) Optimizing DNA ligations for transformations. Focus 8, 30–32.Google Scholar
  17. 17.
    Minks, C., Alefelder, S., Moroder, L., Huber, R., and Budisa, N. (2000) Towards new protein engineering: in vivo building and folding of protein shuttles for drug delivery and targeting by the selective pressure incorporation (SPI) method. Tetrahedron 56, 9431–9442.CrossRefGoogle Scholar
  18. 18.
    Ausubel, F. M. (1987) Current Protocols in molecular Biology. Greene Pub. Associates and Wiley-Interscience, New York.Google Scholar
  19. 19.
    Döring, V., Mootz, H. D., Nangle, L. A., et al. (2001) Enlarging the amino acid set of Escherichia coli by infiltration of the valine coding pathway. Science 292, 501–504.PubMedCrossRefGoogle Scholar
  20. 20.
    Wang, L., Brock, A., Herberich, B., and Schultz, P. G. (2001) Expanding the genetic code of Escherichia coli. Science 292, 498–500.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc. 2007

Authors and Affiliations

  • Jamie M. Bacher
    • 1
  • Andrew D. Ellington
    • 2
    • 3
  1. 1.The Skaggs Institute for Chemical BiologyThe Scripps Research InstituteLa Jolla
  2. 2.Institute for Cellular and Molecular BiologyUniversity of TexasAustin
  3. 3.Department of BiochemistryUniversity of TexasAustin

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