Pharmaceutical Proteins From Methylotrophic Yeasts

  • Eric C. de Bruin
  • Erwin H. Duitman
  • Arjo L. de Boer
  • Marten Veenhuis
  • Ineke G. A. Bos
  • C. Erik Hack
Part of the Methods in Molecular Biology™ book series (MIMB, volume 308)


Because of their favorable properties, methylotrophic yeasts have become increasingly important as cell factories for the production of biomaterials, therapeutic proteins, and vaccines. As a eukaryote, yeast can perform most of the posttranslational modifications that are required to ensure the functionality and/or stability of recombinant human proteins, such as N- and O-linked glycosylation, phosphorylation, and formation of disulfide bonds. In contrast to other yeast systems, foreign genes can be expressed at high levels under control of strong inducible promoters derived from genes encoding proteins that are involved in methanol metabolism. Furthermore, heterologous proteins can be secreted at high levels into the culture medium, which, in combination with the fact that few endogenous proteins are secreted, significantly facilitates purification of the desired protein. Finally, as unicellular microorganisms, methylotrophic yeasts have major advantages in industrial fermentation.


Heterologous Protein Heterologous Gene Methylotrophic Yeast AOX1 Gene Recombinant Human Protein 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


  1. 1.
    Cereghino, J. L. and Cregg, J. M. (2000) Heterologous protein expression in the methylotrophic yeast Pichia pastoris. FEMS Microbiol. Rev. 24, 45–66.PubMedCrossRefGoogle Scholar
  2. 2.
    Gellissen, G. (2000) Heterologous protein production in methylotrophic yeasts. Appl. Microbiol. Biotechnol. 54, 741–750.PubMedCrossRefGoogle Scholar
  3. 3.
    Gleeson, M. A. G., White, C. E., Meininger, D. P., and Komives, E. A. (1998) Generation of protease-deficient strains and their use in heterologous protein expression, in Methods in Molecular Biology, vol. 103: Pichia Protocols (Higgins, D. R. and Gregg, J. M., eds.), Humana, Totowa, NJ, pp. 81–94.CrossRefGoogle Scholar
  4. 4.
    Choi, B. K., Bobrowicz, P., Davidson, R. C., Hamilton, S. R., Kung, D. H., Li, H., et al. (2003) Use of combinatorial genetic libraries to humanize N-linked glycosylation in the yeast Pichia pastoris. Proc. Natl. Acad. Sci. USA 100, 5022–5027.PubMedCrossRefGoogle Scholar
  5. 5.
    Kim, M. W., Rhee, S. K., Kim, J. Y., Shimma, Y., Chiba, Y., Jigami, Y., and Kang H. A. (2004) Characterization of N-linked oligosaccharides assembled on secretory recombinant glucose oxidase and cell wall mannoproteins from the methylotrophic yeast Hansenula polymorpha. Glycobiology 14, 243–251.PubMedCrossRefGoogle Scholar
  6. 6.
    Hamilton, S. R., Bobrowicz, P., Bobrowicz, B., Davidson, R. C., Li, H., Mitchell, T., et al. (2003) Production of complex human glycoproteins in yeast. Science 301, 1244–1246.PubMedCrossRefGoogle Scholar
  7. 7.
    Gleeson, M. A. G. and Sudbery, P. E. (1988) Genetic analysis in the methylotrophic yeast Hansenula polymorpha. Yeast 4, 293–303.CrossRefGoogle Scholar
  8. 8.
    Gietl, C., Faber, K. N., van der Klei, I. J., and Veenhuis, M. (1994) Mutational analysis of the N-terminal topogenic signal of watermelon glyoxysomal malate dehydrogenase using the heterologous host Hansenula polymorpha. Proc. Natl. Acad. Sci. USA 91, 3151–3155.PubMedCrossRefGoogle Scholar
  9. 9.
    Cregg, J. M., Madden, K. R., Barringer, K. J., Thill, G. P., and Stillman, C. A. (1989) Functional characterization of the two alcohol oxidase genes from the yeast Pichia pastoris. Mol. Cell. Biol. 9, 1316–1323.PubMedGoogle Scholar
  10. 10.
    Gatzke, R., Weydemann, U., Janowicz, Z. A., and Hollenberg, C. P. (1995) Stable multicopy integration of vector sequences in Hansenula polymorpha. Appl. Microbiol. Biotechnol. 43, 844–849.PubMedCrossRefGoogle Scholar
  11. 11.
    Becker, D. M. and Guarente L. (1991) High-efficiency transformation of yeast by electroporation. Methods Enzymol. 194, 182–187.PubMedCrossRefGoogle Scholar
  12. 12.
    Faber, K. N, Haima, P., Harder, W., Veenhuis, M., and AB, G. (1994) Highly-efficient electrotransformation of the yeast Hansenula polymorpha. Curr. Genet. 25, 305–310.PubMedCrossRefGoogle Scholar
  13. 13.
    Wu, S. and Letchworth, G. J. (2004) High efficiency transformation by electroporation of Pichia pastoris pretreated with lithium acetate and dithiothreitol. Biotechniques 36, 152–154.PubMedGoogle Scholar
  14. 14.
    Bos, I. G., de Bruin, E. C., Karuntu, Y. A., Modderman, P. W., Eldering, E., and Hack, C. E. (2003) Recombinant human C1-inhibitor produced in Pichia pastoris has the same inhibitory capacity as plasma C1-inhibitor. Biochim. Biophys. Acta 1648, 75–83.PubMedGoogle Scholar
  15. 15.
    Pichia Fermentation Process Guidelines, version B, Invitrogen, Carlsbad, CA.Google Scholar
  16. 16.
    Stratton, J., Chiruvolu, V., and Meagher, M. (1998) High cell-density fermentation, in Methods in Molecular Biology, vol. 103: Pichia Protocols (Higgins, D. R. and Gregg, J. M., eds.), Humana, Totowa, NJ, pp. 107–120.CrossRefGoogle Scholar
  17. 17.
    de Bruin, E. C., de Wolf, F. A., and Laane, N. C. (2000) Expression and secretion of human alpha1(I) procollagen fragment by Hansenula polymorpha as compared to Pichia pastoris. Enzyme Microb. Technol. 26, 640–644.PubMedCrossRefGoogle Scholar
  18. 18.
    Potter, K. J., Zhang, W., Smith, L. A., and Meagher, M. M. (2000) Production and purification of the heavy chain fragment C of botulinum neurotoxin, serotype A, expressed in the methylotrophic yeast Pichia pastoris. Protein Expr. Purif. 19, 393–402.PubMedCrossRefGoogle Scholar
  19. 19.
    Clare, J. J., Romanos, M. A., Rayment, F. B., Rowedder, J. E., Smith, M. A., Payne, M. M., et al. (1991) Production of mouse epidermal growth factor in yeast: high-level secretion using Pichia pastoris strains containing multiple gene copies. Gene 105, 205–212.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc. 2005

Authors and Affiliations

  • Eric C. de Bruin
    • 1
  • Erwin H. Duitman
    • 2
  • Arjo L. de Boer
    • 2
  • Marten Veenhuis
    • 2
  • Ineke G. A. Bos
    • 1
  • C. Erik Hack
    • 3
  1. 1.Department of ImmunopathologySanquin Research at CLBAmsterdamThe Netherlands
  2. 2.Eukaryotic Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute (GBB)University of GroningenHarenThe Netherlands
  3. 3.Departments of Immunopathology and Clinical Chemistry, Sanquin Research at CLBVU Medical CentreAmsterdamThe Netherlands

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