Abstract
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.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsSpringer Nature is developing a new tool to find and evaluate Protocols. Learn more
References
Cereghino, J. L. and Cregg, J. M. (2000) Heterologous protein expression in the methylotrophic yeast Pichia pastoris. FEMS Microbiol. Rev. 24, 45–66.
Gellissen, G. (2000) Heterologous protein production in methylotrophic yeasts. Appl. Microbiol. Biotechnol. 54, 741–750.
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.
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.
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.
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.
Gleeson, M. A. G. and Sudbery, P. E. (1988) Genetic analysis in the methylotrophic yeast Hansenula polymorpha. Yeast 4, 293–303.
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.
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.
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.
Becker, D. M. and Guarente L. (1991) High-efficiency transformation of yeast by electroporation. Methods Enzymol. 194, 182–187.
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.
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.
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.
Pichia Fermentation Process Guidelines, version B, Invitrogen, Carlsbad, CA.
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.
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.
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.
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.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2005 Humana Press Inc.
About this protocol
Cite this protocol
de Bruin, E.C., Duitman, E.H., de Boer, A.L., Veenhuis, M., Bos, I.G.A., Hack, C.E. (2005). Pharmaceutical Proteins From Methylotrophic Yeasts. In: Smales, C.M., James, D.C. (eds) Therapeutic Proteins. Methods in Molecular Biology™, vol 308. Humana Press. https://doi.org/10.1385/1-59259-922-2:065
Download citation
DOI: https://doi.org/10.1385/1-59259-922-2:065
Publisher Name: Humana Press
Print ISBN: 978-1-58829-390-9
Online ISBN: 978-1-59259-922-6
eBook Packages: Springer Protocols