Skip to main content
SpringerLink
Log in
Book cover

Protein Synthesis pp 43–73Cite as

A Biosynthetic Approach for the Incorporation of Unnatural Amino Acids into Proteins

  • Protocol

Part of the book series: Methods in Molecular Biology ((MIMB,volume 77))

Abstract

Unnatural amino acids can be incorporated into proteins or peptides by a variety of existing methods. Peptide synthesis (1–5) and semisynthetic methods (6–10) have been used to substitute novel amino acids into small proteins (<10 kDa) and peptides (11). Solvent accessible, reactive amino acid side chains have also been modified by chemical means (12–18). Modified amino acids have been uniformly incorporated into peptides and proteins with functional analogs of aminoacyl transfer RNAs (tRNAs) (19–21). Furthermore, several unnatural amino acids have been incorporated into dipeptides through the use of chemically misacylated tRNAs (22,23). However, these methods all suffer either from nonselective introduction of the novel amino acid or from size restrictions on the protein of interest.

This is a preview of subscription content, log in via an institution.

Protocol
USD   49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD   129.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD   169.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD   169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Learn about institutional subscriptions

Springer Nature is developing a new tool to find and evaluate Protocols. Learn more

References

  1. Hofmann, K. and Bohn, H. (1966) Studies on polypeptides XXXVI. The effect of pyrazole-imidazole replacements on the s-protein activating potency of an s-peptide fragment. J. Am. Chem. Soc. 88, 5914–5919.

    Article  PubMed  CAS  Google Scholar 

  2. Nakatsuka, T., Sasaki, T., and Kaiser, E. T. (1987) Peptide segment coupling catalyzed by the semisynthetic enzyme thiosubtilisin. J. Am. Chem. Soc. 109, 3808–3810.

    Article  CAS  Google Scholar 

  3. Richards, F. M. and Wyckoff, H. W. (1971) Bovine pancreatic ribonuclease, in The Enzymes (P. D. Boyer, ed.), Academic, New York, pp 647–806.

    Google Scholar 

  4. Kaiser, E. T. (1989) Synthetic approaches to biologically active peptides and proteins including enzymes. Acc. Chem. Res. 22, 47–54.

    Article  CAS  Google Scholar 

  5. Bayer, E. (1991) Towards the chemical synthesis of proteins. Ang. Chem. Int. Ed. Engl. 30, 113–216.

    Article  Google Scholar 

  6. Borras, F. and Offord, R. E. (1970) Protected intermediate for the preparation of semisynthetic insulins. Nature 227, 716–718.

    Article  PubMed  CAS  Google Scholar 

  7. Sealock, R. W. and Laskowski, M. (1969) Enzymatic replacement of the arginyl by a lysyl residue in the reactive site of soybean trypsin inhibitor. Biochem. 8, 3703–3710.

    Article  CAS  Google Scholar 

  8. Inouye, K., Watanabe, K., Morihara, K., Tochino, Y., Kanaya, T., Emura, J., and Sakukibara, S. (1979) Enzyme-assisted semisynthesis of human insulin. J. Am. Chem. Soc. 101, 751–752.

    Article  CAS  Google Scholar 

  9. Offord, R. E. (1987) Protein engineering by chemical means? Prot. Eng. 1(3), 151–157.

    Google Scholar 

  10. Chaiken, I. M. (1981) Semisynthetic peptides and proteins. CRC Crit. Rev. Biochem. 11, 255–301.

    Article  PubMed  CAS  Google Scholar 

  11. Dawson, P. E., Muir, T. W., Clark-Lewis, I., and Kent, S. B. H. (1994) Synthesis of proteins by native chemical ligation. Science 266, 776–779.

    Article  PubMed  CAS  Google Scholar 

  12. Kaiser, E. T. and Lawrence, D. S. (1984) Chemical mutation of enzyme active sites. Science 226, 505–511.

    Article  PubMed  CAS  Google Scholar 

  13. Kaiser, E. T. Lawrence, D. S., and Rokita, S. E. (1985) The chemical modification of enzymatic specificity. Annu. Rev. Biochem. 54, 565–595.

    Article  PubMed  CAS  Google Scholar 

  14. Neet, K. E., Nanci, A., and Koshland, D. E. (1968) Properties of thiol-subtilisin. J. Biol. Chem. 243, 6392–6401.

    PubMed  CAS  Google Scholar 

  15. Polgar, L. and Bender, M. L. (1966) A new enzyme containing a synthetically formed active site. Thiol-subtilisin. J. Am. Chem Soc. 88, 3153–3154.

    Article  CAS  Google Scholar 

  16. Pollack, S. J., Nakayama, G., and Schultz, P. G. (1988) Introduction of nucleophiles and spectroscopic probes into antibody combining sites. Science 242, 1038–1040.

    Article  PubMed  CAS  Google Scholar 

  17. Corey, D. R. and Schultz, P. G. (1987) Generation of a hybrid sequence-specific single-stranded deoxyribonuclease. Science 233, 1401–1403.

    Article  Google Scholar 

  18. Neet, K. E. and Koshland, D. E. (1966) The conversion of serine at the active site of subtilisin to Cysteine a “Chemical Mutation”. Proc. Natl. Acad. Sci. USA 56, 1606–1611.

    Article  PubMed  CAS  Google Scholar 

  19. Krieg, U. C., Walter, P., and Johnson, A. E. (1986) Photocrosslinking of the signal sequence of nascent preprolactin to the 54-Kilodalton polypeptide of the signal recognition particle. Proc. Natl. Acad. Sci. USA 83, 8604–8608.

    Article  PubMed  CAS  Google Scholar 

  20. Brunner, J. (1993) New photolabeling and crosslinking methods. Annu. Rev. Biochem. 62, 483–514.

    Article  PubMed  CAS  Google Scholar 

  21. Baldini, G., Martoglio, B., Schachenmann, A., Zugliani, C., and Brunner, J. (1988) Mischarging E. coli tRNAPhe with L-4′-[3-(tTrifluoromethyl)-3H-diazirin-3-y1]phenylalanine, a photoactivatable analogue of phenylalanine. Biochem. 27, 7951–7959.

    Article  CAS  Google Scholar 

  22. Heckler, T. G., Zama, Y., Naka, T., and Hecht, S. M. (1983) Dipeptide formation with misacylated tRNAPhes. J. Biol. Chem. 258, 4492–4495.

    PubMed  CAS  Google Scholar 

  23. Roesser, J. R., Chorghade, M. S., and Hecht, S. M. (1986) Ribosome-catalyzed formation of an abnormal peptide analogue. Biochem. 25, 6361–6365.

    Article  CAS  Google Scholar 

  24. Cornish, V. W. and Schultz, P. G. (1994) A new tool for studying protein structure and function. Cur. Opin. Struct. Biol. 4, 601–607.

    Article  CAS  Google Scholar 

  25. Ellman, J. A., Mendel, D., Anthony-Cahill, S., Noren, C. J., and Schultz, P. G. (1992) Biosynthetic method for introducing unnatural amino acids site-specifically into proteins. Meth. Enzymol. 202, 301–336.

    Article  Google Scholar 

  26. Noren, C. J., Anthony-Cahill, S. J., Griffith, M. C., and Schultz, P. G. (1989) A general method for site-specific incorporation of unnatural amino acids into proteins. Science 244, 182–188.

    Article  PubMed  CAS  Google Scholar 

  27. Sayers, J. R., Schmidt, W., and Eckstein, F. (1988) 5′-3′ exonuclease in phosphorothioate-based oligonucleotide-directed mutagenesis. Nucleic Acids Res. 16, 791–802.

    Article  PubMed  CAS  Google Scholar 

  28. Barn, J. D., Diala, E. S., Glabe, C. G., Wacker, A., Lyttle, M. H., Dix, T. A., and Chamberlin, R. A. (1991) Site-specific incorporation of nonnatural residues during in vitro protein biosynthesis with semisynthetic aminoacyl-tRNAs. Biochemistry 30, 5411–5421.

    Article  Google Scholar 

  29. Bam, J. D., Switzer, C., Chamberlin, R. A., and Benner, S. (1992) Ribosome-mediated incorporation of non-standard amino acids into a peptide through expansion of the genetic code. Nature 356, 537–539.

    Article  Google Scholar 

  30. Cornish, V. W., Mendel, D., and Schultz, P. G. (1995) Probing protein structure and function with an expanded genetic code. Ang. Chem. Int. Ed. Engl. 34, 621–633.

    Article  CAS  Google Scholar 

  31. Sambrook, J., Fritsch, E. F., and Maniatis, T., eds. (1989) Molecular Cloning: A Laboratory Manual Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.

    Google Scholar 

  32. Williams, R. M. (1989) Synthesis of optically active α-amino acids. Pergamon, New York.

    Google Scholar 

  33. Roberts, D. C. and Vellaccio, F. (1983) Unusual amino acids in peptide synthesis. Peptides 5, 341–449.

    CAS  Google Scholar 

  34. Seebach, D., Juaristi, E., Miller, D. D., Schickli, C., and Weber, T. (1987) Addition of chiral glycine, methionine, and vinylglycine enolate derivatives to aldehydes and ketones in the preparation of enantiomericallypure α-amino-β-hydroxy acids. Helv. Chim. Acta. 70, 237–261.

    Article  CAS  Google Scholar 

  35. Williams, R. M. and Im, M.-N. (1988) Asymmetric synthesis of α-amino acids comparison of enolate vs cation functionalization on n-boc-5,6-diphenyl-2,3,5,6-tetrahydro-4H-1,4-oxazin-2-ones. Tet. Lett. 29, 6075–6078.

    Article  CAS  Google Scholar 

  36. O’Donnell, M. J., Bennett, W. D., and Wu, S. (1989) The stereoselective synthesis of α-amino acids by phase-transfer catalysis. J. Am. Chem. Soc. 111, 2353–2355.

    Article  Google Scholar 

  37. Evans, D. A., Sjogren, E. B., Weber, A. E., and Conn, R. E. (1987) Asymmetric synthesis of anti-β-hydroxy-α-amino acids. Tet. Lett. 28, 39–42.

    Article  CAS  Google Scholar 

  38. Mendel, D., Ellman, J. A., and Schultz, P. G. unpublished results.

    Google Scholar 

  39. Amit, B., Zehavi, U., and Patchornik, A. (1974) Photosensitive protecting groups of amino sugars and their use in glycoside synthesis 2-nitrobenzyloxy-carbonylamino and 6-nitroveratryloxycarbonylamino derivatives. J. Org. Chem. 39, 192–196.

    Article  Google Scholar 

  40. Greene, T. W. and Wuts, P. G. M. (1991) Protective Groups in Organic Synthesis. Wiley, New York.

    Google Scholar 

  41. Jonin, P., Castro, B., Zeggaf, C., Pantaloni, A., Senet, J. P., Lecolier, S., and Sennyey, G. (1987) Mono-esterification of N-Protected diacids aspartic and glutamic by chloroformate activation. Tet. Lett. 28, 1665–1668.

    Article  Google Scholar 

  42. Robertson, S. A., Ellman, J. A., and Schultz, P. G. (1991) A general and efficient route for chemical aminoacylation of transfer RNAs. J. Am. Chem. Soc. 113, 2722–2729.

    Article  CAS  Google Scholar 

  43. Uhlmann, E. and Engels, J. (1986) Chemical 5′-phosphorylation of oligonucleotides valuable in automated DNA synthesis. Tet. Lett. 27, 1023–1026.

    Article  CAS  Google Scholar 

  44. Zawadzki, V. and Gross, H. J. (1991) Rapid and simple purification of T7 RNA polymerase. Nucleic Acids Res. 19, 1948.

    Article  PubMed  CAS  Google Scholar 

  45. Davanloo, P., Rosenberg, A. H., Dunn, J. J., and Studier, F. W. (1984) Cloning and expression of the gene for bacteriophage T7 RNA polymerase. Proc. Natl. Acad. Sci. USA 81, 2035–2039.

    Article  PubMed  CAS  Google Scholar 

  46. Burgess, R. R. and Jendrisak, J. J. (1975) A procedure for the rapid, large-scale purification of E. coli DNA-dependent RNA polymerase involving polymin p precipitation and DNA-cellulose chromatography. Biochemistry 14, 4634–4638.

    Article  PubMed  CAS  Google Scholar 

  47. Zubay, G. (1973) In vitro synthesis of protein in microbial systems. Annu. Rev. Genet. 7, 267–287.

    Article  PubMed  CAS  Google Scholar 

  48. Pratt, J. (1984) in Transcription and Translation: A Practical Approach (Hames, B. D and Higgins, S. J., eds.), IRL, Oxford, pp. 179–209.

    Google Scholar 

  49. Scopes, R. (1982) Protein Purification Principles and Practice. Springer-Verlag, New York.

    Google Scholar 

  50. Deutscher, M. P., ed. (1990) Guide to Protein Purification. Academic, San Diego.

    Google Scholar 

  51. Bedouelle, H. and Duplay, P. (1988) Production in E. coli and one-step purification of bifunctional hybrid proteins which bind maltose. Eur. J. Biochem. 171, 541–549.

    Article  PubMed  CAS  Google Scholar 

  52. Smith, D. B. and Johnson, K. S. (1988) Single-step purification of polypeptides expressed in E. coli as fusions with glutathione S-transferase. Gene 67, 31–40.

    Article  PubMed  CAS  Google Scholar 

  53. Crowe, J., Dobeli, H., Gentz, R., Hochuli, E., Stuber, D., and Denco, K. (1994) 6xHis-Ni-NTA chromatography as a superior technique in recombinant protein expression/purification. Meth. Mol. Biol. 31, 371–387.

    CAS  Google Scholar 

  54. Scharf, S. J., Horn, G. T., and Erlich, H. A. (1986) Direct cloning and sequence analysis of enzymatically amplified genomic sequences. Science 233, 1076–1078.

    Article  PubMed  CAS  Google Scholar 

  55. Chladek, S. and Sprinzl, M. (1985) The 3′-End of tRNA and its role in protein biosynthesis. Angew. Chem. Int. Ed. Engl. 24, 371–391.

    Article  Google Scholar 

  56. Hecht, S. M. (1992) Probing the synthetic capabilities of a center of biochemical catalysts. Acc. Chem. Res. 25(12), 545–552.

    Article  Google Scholar 

  57. Suich, D. (1993) Mutagenesis of the Recognition Helix of 434 Repressor with Nonproteinogenic Amino Acids, Ph. D. Thesis. University of California, Berkeley.

    Google Scholar 

  58. Mendel, D., Ellman, J., and Schultz, P. G. (1993) Protein biosynthesis with conformationally restricted amino acids. J. Am. Chem. Soc. 115, 4359–4360.

    Article  CAS  Google Scholar 

  59. Cornish, V. W., Benson, D. R., Altenbach, C. A., Hideg, K., Hubbell, W. L., and Schultz, P. G. (1994) Site-specific incorporation of biophysical probes into proteins. Proc. Natl. Acad. Sci. USA 91, 2910–2914.

    Article  PubMed  CAS  Google Scholar 

  60. Louie, A. and Jurnak, F. (1984) Kinetic studies of E. coli elongation factor tuguanosine 5′-triphosphate-aminoacyl-tRNA complexes. Biochemistry 24, 6433–6439.

    Article  Google Scholar 

  61. Bhuta, A., Quiggle, K., Ott, T., Ringer, D., and Chladek, S. (1981) Stereochemical control of ribosomal peptidyltransferase reaction. Role of amino acid side-chain orientation of acceptor substrate. Biochemistry 20, 8–15.

    Article  PubMed  CAS  Google Scholar 

  62. Bhuta, P., Kumar, G., and Chladek, S. (1982) Elongation factor tu-ribosome Dependent guanosine 5′-triphosphate hydrolysis, elucidation of the role of the aminoacyl transfer ribonucleic acid 3′-terminus and site(s) involved in the inducing of the guanosinetriphosphatase reaction. Biochemistry 21, 899–905.

    Article  PubMed  CAS  Google Scholar 

  63. Quiggle, K., Kumar, G., Oh, T. W., Ryu, E. K., and Chladek, S. (1981) Donor site of ribosomal peptidyltransferase: investigation of substrate specificity using 2′(3′)-O-(N-Acylaminoacyl)-dinucleoside phosphates as models of the 3′-terminus of N-Acylaminoacyl transfer ribonucleic acid. Biochemistry 20, 3480–3485.

    Article  PubMed  CAS  Google Scholar 

  64. Fahnestock, S., Neumann, H., Shashoua, V., and Rich, A. (1970) Ribosome-catalyzed ester formation. Biochemistry 9, 2477–2483.

    Article  PubMed  CAS  Google Scholar 

  65. Ellman, J. A., Mendel, D., and Schultz, P. G. (1992) Site-specific incorporation of novel backbone structures into proteins. Science 255, 197–200.

    Article  PubMed  CAS  Google Scholar 

  66. Grant, M. M., Brown, A. S., Corwin, L. M., Troxler, R. F., and Franzblau, C. (1975) Effect of L-azetidine 2-carboxylic acid on growth and proline metabolism in E. Coli. Biochim. Biophys. Acta. 404, 180–187.

    Article  PubMed  CAS  Google Scholar 

  67. Bain, J. D., Wacker, D. A., Kuo, E. E., and Chamberlin, A. R. (1991) Site-specific incorporation of nonnatural residues into peptides effect of residue structure on suppression and translational efficiencies. Tetrahedron 47, 2389–2400.

    Article  CAS  Google Scholar 

  68. Benson, D., Ellman, J. A., and Schultz, P. G. unpublished results.

    Google Scholar 

  69. Baldini, G., Martoglio, B., Schachenmann, A., Zugliani, C., and Brunner, J. (1988) Mischarging E. coli tRNAPhe with L-4′-[3-(trifluoromethyl)-3H-diazirin-3-yl]phenylalanine, a photoactivatable analogue of phenylalanine. Biochemistry 27, 7951–7959.

    Article  PubMed  CAS  Google Scholar 

  70. Ayer, D. and Yarus, M. (1986) The context effect does not require a fourth base pair. Science 231, 393–395.

    Article  PubMed  CAS  Google Scholar 

  71. Bossi, L. (1983) Translation of UAG codon by suppressor tRNA is Affected by the sequence following UAG in the message. J. Mol. Biol. 164, 73–87.

    Article  PubMed  CAS  Google Scholar 

  72. Miller, A. and Albertini, A. (1983) Effects of surrounding sequence on the suppression of nonsense codons. J. Mol. Biol. 164, 59–71.

    Article  PubMed  CAS  Google Scholar 

  73. Hames, B. D. and Higgins, S. J. (1984) Transcription and Translation: A Practical Approach. IRL, Oxford.

    Google Scholar 

  74. Spirin, A. S., Baranov, V. I., Ryabova, L. A., Ovodov, S. Y., and Alahhov, Y. B. (1988) A continuous cell-free translation system capable of producing polypeptides in high yield. Science 242, 1162–1164.

    Article  PubMed  CAS  Google Scholar 

  75. Landman, O. and Spiegelman, S. (1955) Enzyme formation in protoplasts of Bacillus megaterium. Proc. Natl. Acad. Sci. USA 41, 698–704.

    Article  PubMed  CAS  Google Scholar 

  76. Nomura, M., Hosoda, J., and Nishimura, S. (1958) Enzyme formation in lysozyme lysate of Bacillus subtilis. Biochim. Biophys. Acta. 29, 161–167.

    Article  PubMed  CAS  Google Scholar 

  77. Kleina, L. G., Masson, J. M., Normanly, J., Abelson, J., and Miller, J. H. (1990) Construction of E. coli amber suppressor tRNA genes. II. Synthesis of additional tRNA genes and improvement of suppressor efficiency. J. Mol. Biol. 213, 705–718.

    Article  PubMed  CAS  Google Scholar 

  78. Cornish, V. W. and Schultz, P. G. unpublished results.

    Google Scholar 

  79. Mendel, D. and Schultz, P. G. unpublished results.

    Google Scholar 

  80. Chunhua, M., Kudlicki, W., Odom, O. W., Kramer, G., and Hardesty, B. (1993) In vitro protein engineering using synthetic tRNAAla with different anticodons. Biochemistry 32, 7937–7945.

    Google Scholar 

  81. Nowak, M. W., Kearney, P. C., Sampson, J. R., Saks, M. E., Labarca, C. G., Silverman, S., K., Zhong, W., Thorson, J. S., Abelson, J. N., Davidson, N., Schultz, P. G., Dougherty, D. A., and Lester, H. A. (1995) Nicotinic receptor binding site probed with unnatural amino acid incorporation in intact cells. Science 268, 439–442.

    Article  PubMed  CAS  Google Scholar 

  82. Nevin, D. and Pratt, J. (1991) A coupled in vitro transcription-translation system for the exclusive synthesis of polypeptides expressed from the T7 promoter. FEBS Lett. 291, 259–263.

    Article  PubMed  CAS  Google Scholar 

  83. Lesley, S. A. (1995) Preparation and use of E. coli S-30 extracts. Meth. Mol. Biol. 37, 265–278.

    CAS  Google Scholar 

  84. Noren, C. J. (1990) Site-specific Mutagenesis with Unnatural Amino Acids, Ph. D. Thesis. University of California, Berkeley.

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Chemistry, Howard Hughes Medical Institute, University of California, Berkeley, CA

    Jon S. Thorson, Virginia W. Cornish, Jeannie E. Barrett, Sharon T. Cload, Takato Yano & Peter G. Schultz

Authors
  1. Jon S. Thorson
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Virginia W. Cornish
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jeannie E. Barrett
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Sharon T. Cload
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Takato Yano
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Peter G. Schultz
    View author publications

    You can also search for this author in PubMed Google Scholar

Editor information

Editors and Affiliations

  1. Blond McIndoe Center, Queen Victoria Hospital, Sussex, England

    Robin Martin

Rights and permissions

Reprints and permissions

Copyright information

© 1998 Humana Press Inc.

About this protocol

Cite this protocol

Thorson, J.S., Cornish, V.W., Barrett, J.E., Cload, S.T., Yano, T., Schultz, P.G. (1998). A Biosynthetic Approach for the Incorporation of Unnatural Amino Acids into Proteins. In: Martin, R. (eds) Protein Synthesis. Methods in Molecular Biology, vol 77. Springer, Totowa, NJ. https://doi.org/10.1385/0-89603-397-X:43

Download citation

  • DOI: https://doi.org/10.1385/0-89603-397-X:43

  • Publisher Name: Springer, Totowa, NJ

  • Print ISBN: 978-0-89603-397-9

  • Online ISBN: 978-1-59259-563-1

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation