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Towards Engineering Proteins by Site-Directed Incorporation In Vivo of Non-Natural Amino Acids

Bio/Technology volume 12, pages 678–682 (1994)Cite this article

Abstract

Altering protein structure via the techniques of protein engineering has already allowed the development of proteins displaying both modified and novel activities. The only limitation of conventional site-directed mutagenesis, the cornerstone of protein engineering, is that substitutions are restricted to the 20 naturally occurring, proteinogenic amino acids. However, the discovery of a 21st amino acid, selenocysteine, and the development of novel in vitro translation systems have demonstrated that considerably more substitutions are possible. To this end, a number of experimental approaches have been developed that allow the incorporation of synthetic amino acids into proteins. Some of these have already been successfully applied in vitro and efforts to transfer this technology to in vivo systems are now underway.

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References

  1. Smith, M.S. 1985. In vitro mutagenesis. Annu. Rev. Genet. 19: 423–462.

    Article  CAS  PubMed  Google Scholar 

  2. Medynski, D. 1992. Genetic approaches to protein structure and function: point mutations as modifiers of protein function. Bio/Technology 10: 1002–1006.

    CAS  Google Scholar 

  3. Creighton, T.E. 1993. Proteins: structures and molecular properties, 2nd Ed. W.H. Freeman and Company, New York.

    Google Scholar 

  4. Matthews, B.W. 1993. Structural and genetic analysis of protein stability. Annu. Rev. Biochem. 62: 139–160.

    Article  CAS  PubMed  Google Scholar 

  5. Böck, A., Forchhammer, K., Heider, J., Leinfelder, W., Sawers, G., Veprek, B. and Zinoni, F. 1991. Selenocysteine: the 21st amino acid. Mol. Microbiol. 5: 515–520.

    Article  PubMed  Google Scholar 

  6. Noren, C.J., Anthony-Cahill, S.J., Griffith, M.C. and Schulte, P.G. 1989. A general method for site-specific incoiporation of unnatural amino acids into proteins. Science 244: 182–188.

    Article  CAS  PubMed  Google Scholar 

  7. Mazel, D., Pochet, S. and Marlière, P. 1994. Genetic characterization of polypeptide defomiylase, a distinctive enzyme of eubacterial translation. EMBO J. 13: 914–923.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Rajbhandary, U.L. 1994. Initiator transfer RNAs. J. Bacteriol. 176: 547–552.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Zinoni, P., Birkmann, A., Leinfelder, W. and Böck, A. 1987. Cotranslational insertion of selenocysteine into formate dehydrogenase from Escherichia coli directed by a UGA codon. Proc. Natl. Acad. Sci. USA 84: 3156–3160.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Böck, A., Forchhammer, K., Heider, J. and Baron, C. 1991. Selenoprotein synthesis: an expansion of the genetic code. Trends Biochem. Sci. 16: 463–467.

    Article  PubMed  Google Scholar 

  11. Ringquist, S., Schneider, D., Gibson, T., Baron, C., Böck, A. and Gold, L. 1994. Recognition of the mRNA selenocysteine insertion sequence by the specialized translation elongation factor SELB. Genes Dev. 8: 376–385.

    Article  CAS  PubMed  Google Scholar 

  12. Hendrickson, W.A., Horton, J.R. and LeMaster, D.M. 1990. Selenomethionyl proteins produced for analysis by multiwavelength anomalous diffraction (MAD): a vehicle for direct determination of three-dimensional structure. EMBO J. 9: 1665–1672.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Hendrickson, W.A. 1991. Determination of macromolecular structures from anomalous diffraction of synchrotron radiation. Science 254: 51–58.

    Article  CAS  PubMed  Google Scholar 

  14. Müller, S., Senn, H., Gsell, B., Vetter, W., Baron, C. and Böck, A. 1994. The formation of diselenide bridges in proteins by incorporation of selenocysteine residues–Biosynthesis and characterization of (Se) (2)-thioredoxin. Biochemistry 33: 3404–3412.

    Article  PubMed  Google Scholar 

  15. Hilvert, D. 1991. Extending the chemistry of enzymes and abzymes. Trends Biotechnol. 9: 11–17.

    Article  CAS  PubMed  Google Scholar 

  16. Bell, I.M. and Hilvert, D. 1993. Peroxide dependence of the semisynthetic enzyme selenosubtilisin. Biochemistry 32: 13969–13973.

    Article  CAS  PubMed  Google Scholar 

  17. Mottagui-Tabar, S., Bjornsson, A. and Isaksson, L.A. 1994. The second to last amino acid in the nascent peptide as a codon context determinant. EMBO J. 13: 249–257.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Hatfield, D. and Diamond, A. 1993. UGA: a split personality in the genetic code. Trends Genet. 9: 69–70.

    Article  CAS  PubMed  Google Scholar 

  19. Osawa, S., Jukes, T.H., Watanabe, K. and Muto, A. 1992. Recent evidence for evolution of the genetic code. Microbiol. Rev. 56: 229–264.

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Richmond, M.H. 1962. The effect of amino acid analogues on growth and protein synthesis in microorganisms. Bacteriol. Rev. 26: 398–420.

    CAS  PubMed  PubMed Central  Google Scholar 

  21. Tam, R. and Saier, M.H. Jr., 1993. Structural, functional, and evolutionary relationships among extracellular solute-binding receptors of bacteria. Microbiol. Rev. 57: 320–346.

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Reizer, J., Finley, K., Kakuda, D., Macleod, C.L., Reizer, A. and Saier, M.H. Jr., 1993. Mammalian integral membrane receptors are homologous to facilitators and antiporters of yeast, funghi, and eubacteria. Protein Sci. 2: 20–30.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Hennecke, H. and Böck, A. 1975. Altered α subunits in phenylalanyl-tRNA synthetases from p-fluorophenylalanine-resistant strains of Escherichia coli. Eur. J. Biochem. 55: 431–437.

    Article  CAS  PubMed  Google Scholar 

  24. Jakubowski, H. and Goldman, E. 1992. Editing of errors in selection of amino acids for protein synthesis. Microbiol. Rev. 56: 412–429.

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Kim, H.Y., Ghosh, G., Schulman, L.H., Brunie, S. and Jakubowski, H. 1993. The relationship between synthetic and editing functions of the active site of an aminoacyl-tRNA synthetase. Proc. Natl. Acad. Sci. USA 90: 11553–11557.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Thompson, R.C. 1988. EFTu provides an internal kinetic standard for translational accuracy. Trends Biochem. Sci. 13: 91–93.

    Article  CAS  PubMed  Google Scholar 

  27. Stanzel, M., Schön, A. and Sprinzl, M. 1994. Discrimination against misacylated tRNA by chloroplast elongation factor Tu. Eur. J. Biochem. 219: 435–439.

    Article  CAS  PubMed  Google Scholar 

  28. Ibba, M., Kast, P. and Hennecke, H. 1994. Substrate specificity is determined by amino acid binding pocket size in Escherichia coli phenylalanyl-tRNA synthetase. Biochemistry. In press.

  29. Hortin, G. and Boime, I. 1983. Applications of amino acid analogs for studying co-and posttranslational modifications of proteins. Methods Enzymol. 96: 777–784.

    Article  CAS  PubMed  Google Scholar 

  30. Wilson, M.J. and Hatfield, D.L. 1984. Incorporation of modified amino acids into proteins in vivo. Biochim. Biophys. Acta 781: 205–215.

    Article  CAS  PubMed  Google Scholar 

  31. Heinemeyer, W., Kleinschmidt, J.A., Saidowsky, J., Escher, C. and Wolf, D.H. 1991. Proteinase yscE, the yeast proteosome/multicatalytic-multifunctional proteinase: mutants unravel its function in stress-induced proteolysis and uncover its necessity for survival. EMBO J. 10: 555–562.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Kohno, T., Kohda, D., Haruki, M., Yokoyama, S. and Miyazawa, T. 1990. Nonprotein amino acid furanomycin, unlike isoleucine in chemical structure, is charged to isoleucine tRNA by isoleucyl-tRNA synthetase and incorporated into protein. J. Biol. Chem. 265: 6931–6935.

    CAS  PubMed  Google Scholar 

  33. Lemeignan, B., Sonigo, P. and Marlière, P. 1993. Phenotypic suppression by incorporation of an alien amino acid. J. Mol. Biol. 231: 161–166.

    Article  CAS  PubMed  Google Scholar 

  34. Koide, H., Yokoyama, S., Kawai, G., Ha, J-M., Oka, T., Kawai, S., Miyake, T., Fuwa, T. and Miyazawa, T. 1988. Biosynthesis of a protein containing a nonprotein amino acid by Escherichia coli: L-2 amino hexanoic acid at position 21 in human epidermal growth factor. Proc. Natl. Acad. Sci. USA 85: 6237–6241.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Hinds, M.G., King, R.W. and Feeney, J. 1992. 19F n.m.r. studies of conformational changes accompanying cyclic AMP binding to 3-fluorophenylalanine-containing cyclic AMP receptor protein from Escherichia coli. Biochem. J. 287: 627–632.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Kanemori, M., Mori, H. and Yura, T. 1994. Induction of Escherichia coli heat shock proteins by abnormal proteins results solely from stabilization of σ32, p. 142. In: Biology of Heat Shock Proteins and Molecular Chaperones. Proceedings of the 1994 meeting on Cold Spring Harbor Laboratory, Cold Spring Harbor, New York.

    Google Scholar 

  37. Ross, J.B.A., Senear, D.F., Waxman, E., Kombo, B.B., Rusinova, E., Huang, Y.E., Laws, W.R., Ross, J.B.A., and Hasselbacher, C.A. 1992. Spectral enhancement of proteins: biological incorporation and fluorescene characterization of 5-hydroxytryptophan in bacteriophage γ cI represser. Proc. Natl. Acad. Sci. USA 89: 12023–12027.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Holman, C. and Benisek, W.F. 1994. Extent of proton transfer in the transition states of the reaction catalyzed by the Δ5-3-ketosteroid isomerase of Comamonas (Pseudomonas) testosteroni: site-specific replacement of the active site base, aspartate 38, by the weaker base alanine-3-sulfinate. Biochemistry 33: 2672–2681.

    Article  CAS  PubMed  Google Scholar 

  39. Kim, D-W., Yoshimura, T., Esaki, N., Satoh, E. and Soda, K. 1994. Studies of the active-site lysyl residue of thermostable aspartate aminotransferase: combination of site-directed mutagenesis and chemical modification. J. Biochem. (Tokyo) 115: 93–97.

    Article  CAS  PubMed  Google Scholar 

  40. Normanly, J., Kleina, L.G., Masson, J-M., Abelson, J. and Miller, J.H. 1990. Construction of Escherichia coli amber suppressor tRNA genes. J. Mol. Biol. 213: 719–726.

    Article  CAS  PubMed  Google Scholar 

  41. Michaels, M.L., Kim, C.W., Matthews, D.A. and Miller, J.H. 1990. Escherichia coli thymidyiate synthase: amino acid substitutions by suppression of amber nonsense mutations. Proc. Natl. Acad. Sci. USA 87: 3957–3961.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Ellman, J., Mendel, D., Anthony-Cahill, S., Noren, C.J. and Schultz, P.G. 1991. Biosynthetic methods for introducing unnatural amino acids site-specifically into proteins. Methods Enzymol. 202: 301–336.

    Article  CAS  PubMed  Google Scholar 

  43. Brunner, J. 1993. Biosynthetic incorporation of non-natural amino acids into proteins. Chem. Soc. Rev. 22: 183–189.

    Article  CAS  Google Scholar 

  44. 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 

  45. Spirin, A.S., Baranov, V.I., Ryabova, L.A., Ovodov, S.Y. and Alakhov, Y.B. 1988. A continuous cell-free translation system capable of producing poly peptides in high yield. Science 242: 1162–1164.

    Article  CAS  PubMed  Google Scholar 

  46. Ying, W., Zhang, D.Y. and Kramer, F.R. 1992. Amplifiable messenger RNA. Proc. Natl. Acad. Sci. USA 89: 11769–11773.

    Article  Google Scholar 

  47. Sonar, S., Patel, N., Fischer, W. and Rothschild, K.J. 1993. Cell-free synthesis, functional refolding, and spectroscopic characterization of bacteriorhodopsin, an integral membrane protein. Biochemistry 32: 13777–0781.

    Article  CAS  PubMed  Google Scholar 

  48. Brunner, J. 1993. New photolabelling and crosslinking methods. Annu. Rev. Biochem. 62: 483–514.

    Article  CAS  PubMed  Google Scholar 

  49. High, S., Martoglio, B., Görlich, D., Andersen, S.S.L., Ashford, A.J., Giner, A., Hartmann, E., Prehn, S., Rapoport, T.A., Dobberstein, B. and Brunner, J. 1993. Site-specific photocross-linking reveals that sec61p and TRAM contact different regions of a membrane-inserted signal sequence. J. Biol. Chem. 268: 26745–26751.

    CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  51. Mendel, D., Ellman, J.A., Chang, Z., Veenstra, D.L., Kollman, P.A. and Schultz, P.G. 1992. Probing protein stability with unnatural amino acids. Science 256: 1798–1802.

    Article  CAS  PubMed  Google Scholar 

  52. Chung, H.-H., Benson, D.R. and Schultz, P.G. 1993. Probing the structure and mechanism of ras protein with an expanded genetic code. Science 259: 806–809.

    Article  CAS  PubMed  Google Scholar 

  53. Judice, J.K., Gamble, T.R., Murphy, E.C., de Vos, A. M. and Schultz, P.G. 1993. Probing the mechanism of staphylococcal nuclease with unnatural amino acids: kinetic and structural studies. Science 261: 1578–1581.

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  55. Piccirilli, J.A., Krauch, T., Moroney, S.E. and Benner, S.A. 1990. Enzymatic incorporation of a new base pair into DNA and RNA extends the genetic alphabet. Nature 343: 33–37.

    Article  CAS  PubMed  Google Scholar 

  56. Bain, J.D., Switzer, C., Chamberlain, A.R. and Benner, S.A. 1992. Ribo-some-mediated incorporation of a non-standard amino acid into a peptide through expansion of the genetic code. Nature 356: 537–539.

    Article  CAS  PubMed  Google Scholar 

  57. Schon, A., Kannangara, C.G., Gough, S. and Soil, D. 1988. Protein biosynthesis in organelles requires misaminoacylation of tRNA. Nature 331: 187–190.

    Article  CAS  PubMed  Google Scholar 

  58. Carter, C.W., Jr. 1993. Cognition, mechanism, and evolutionary relationships in aminoacyl-tRNA synthetases. Annu. Rev. Biochem. 62: 715–748.

    Article  CAS  PubMed  Google Scholar 

  59. Kast, P. and Hennecke, H. 1991. Amino acid substrate specificity of Escherichia coli phenylalanyl-tRNA synthetase altered by distinct mutations. J. Mol. Biol. 222: 99–124.

    Article  CAS  PubMed  Google Scholar 

  60. Kast, P. 1991. Investigations on Escherichia coli phenylalanyl-tRNA synthetase at the molecular level. Ph.D. Thesis ETH Nr. 9468.

  61. McClain, W.H. 1993. Rules that govern tRNA identity in protein synthesis. J. Mol. Biol. 234: 257–280.

    Article  CAS  PubMed  Google Scholar 

  62. Saks, M.E., Sampson, J.R. and Abelson, J.N. 1994. The transfer RNA identity problem: a search for rules. Science 263: 191–197.

    Article  CAS  PubMed  Google Scholar 

  63. Rould, M.A., Perona, J.J. and Steitz, T.A. 1991. Structural basis of anticodon loop recognition by glutaminyl-tRNA synthetase. Nature 352: 213–218.

    Article  CAS  PubMed  Google Scholar 

  64. Weygand-Durasevic, L., Schwob, E. and Söil, D. 1993. Acceptor end binding domain interactions ensure correct aminoacylation of transfer RNA. Proc. Natl. Acad. Sci. USA 90: 2010–2014.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  65. Jacobsen, J.R., Prudent, J.R., Kochersperger, L., Yonkovich, S. and Schultz, P.G. 1992. An efficient antibody-catalyzed aminoacylation reaction. Science 256: 365–367.

    Article  CAS  PubMed  Google Scholar 

  66. Kast, P. 1994. pKSS-a second-generation general purpose cloning vector for efficient positive selection of recombinant clones. Gene 138: 109–114.

    Article  CAS  PubMed  Google Scholar 

  67. Kurzchalia, T.V., Wiedmann, M., Breter, H., Zimmermann, W., Bauschke, E. and Rapoport, T.A. 1988. tRNA-mediated labelling of proteins with biotin. Eur. J. Biochem. 172: 663–668.

    Article  CAS  PubMed  Google Scholar 

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  1. Mikrobiologisches Institut, Eidgenössische Technische Hochschule, ETH-Zentrum/LFV, Schmelzbergstrasse 7, CH-8092, Zürich, Switzerland

    Michael Ibba & Hauke Hennecke

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Correspondence to Michael Ibba.

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Ibba, M., Hennecke, H. Towards Engineering Proteins by Site-Directed Incorporation In Vivo of Non-Natural Amino Acids. Nat Biotechnol 12, 678–682 (1994). https://doi.org/10.1038/nbt0794-678

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