Skip to main content
SpringerLink
Log in

Isotopic Labeling of Heterologous Proteins in the Yeast Pichia pastoris and Kluyveromyces lactis

  • Protocol
  • First Online:
Protein NMR Techniques

Part of the book series: Methods in Molecular Biology ((MIMB,volume 831))

Abstract

Several protein expression systems are available for the preparation of stable isotope-labeled recombinant proteins for NMR studies. Yeast expression systems have several advantages over prokaryotic systems, such as the widely used Escherichia coli expression system. Protein expression using the methylotrophic yeast Pichia pastoris is commonly employed for the preparation of isotope-labeled proteins. Recently, the hemiascomycete yeast Kluyveromyces lactis expression system was reported as being useful for preparing proteins for NMR studies. Since each yeast expression system has different features, their applications have increased in number. In this chapter, we describe procedures for the efficient production of uniformly isotope-labeled proteins using the P. pastoris and the K. lactis yeast expression systems.

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

Access this chapter

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 89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 119.99
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

Institutional subscriptions

References

  1. Cregg, J. M., Vedvick, T. S., and Raschke, W. C. (1993) Recent advances in the expression of foreign genes in Pichia pastoris. Biotechnology 11, 905–910.

    Google Scholar 

  2. Pickford, A. R., and O’Leary, J. M. (2008) Isotopic labeling of recombinant proteins from the methylotrophic yeast Pichia pastoris, in Methods in Molecular Biology (Downing, A. K., Ed.), vol. 278, pp. 17–33, Humana Press, Totowa, NJ.

    Google Scholar 

  3. Lin Cereghino, G. P., Cereghino, J. L., Ilgen, C., and Cregg, J. M. (2002) Production of recombinant proteins in fermenter cultures of the yeast Pichia pastoris. Curr. Opin. Biotechnol. 13, 329–332.

    Google Scholar 

  4. Daley, R., and Hearn, M. T. (2005) Expression of heterologous proteins in Pichia pastoris: a useful experimental tool in protein engineering and production. J. Mol. Recognit. 18, 119–138.

    Google Scholar 

  5. Cos, O., Ramόn, R., Montesinos, J. L., and Valero F. (2006) Operational strategies, monitoring and control of heterologous protein production in the methylotrophic yeast Pichia pastoris under different promoters: A review. Microbial Cell Factories 5, 17–36.

    Google Scholar 

  6. Takahashi, H., and Shimada, I. (2010) Production of isotopically labeled heterologous proteins in non-E. coli prokaryotic cells. J. Biomol. NMR 46, 3–10.

    Google Scholar 

  7. Invitrogen Corporation. EasySelect™ Pichia Expression Kit: A manual of methods for expression of recombinant proteins using pPICZ and pPICZα in Pichia pastoris, Version I. Available at http://tools.invitrogen.com/content/sfs/manuals/easyselect_man.pdf.

  8. Laroche, Y., Strome, V., De Meutter, J., Messens, J., and Lauwereys, M. (1994) High-level secretion and very efficient isotopic labeling of tick anticoagulant peptide (TAP) expressed in the methylotrophic yeast, Pichia pastoris. Bio/Technol 12, 1119–1124.

    Google Scholar 

  9. Denton, H., Smith, M., Husi, H., Uhrin, D., Barlow, P. N., Batt, C. A., and Sawyer, L. (1998) Isotopically labeled bovine β-lactoglobulin for NMR studies expressed in Pichia pastoris. Protein Expr. Purif. 14, 97–103.

    Google Scholar 

  10. Wood, M. J., and Komives, E. A. (1999) Production of large quantities of isotopically labeled protein in Pichia pastoris by fermentation.. J. Biomol. NMR 13, 149–159.

    Google Scholar 

  11. Mine, S., Ueda, T., Hashimoto, Y., Tanaka, Y., and Imoto, T. (1999) High-level expression of uniformly 15N-labeled hen lysozyme in Pichia pastoris and identification of the site in hen lysozyme where phosphate ion binds using NMR measurements. FEBS Lett. 448, 33–37.

    Google Scholar 

  12. van den Burg, H. A., de Wit, P. J., and Vervoort, J. (2001) Efficient 13C/15N double labeling of the avirulence protein AVR4 in a methanol-utilizing strain (Mut+) of Pichia pastoris. J. Biomol. NMR 20, 251–261.

    Google Scholar 

  13. Rodriguez, E., and Krishna, N. R. (2001) An economical method for 13C/15N isotopic labeling of proteins expressed in Pichia pastoris. J. Biochem. 130, 19–22.

    Google Scholar 

  14. Morgan, W. D., Kragt, A., and Feeney, J. (2000) Expression of deuterium-isotope-labelled protein in the yeast Pichia pastoris for NMR study. J. Biomol. NMR 17, 337–347.

    Google Scholar 

  15. Ichikawa, M., Osawa, M., Nishida, N., Goshima, N., Nomura, N., and Shimada, I. (2007) Structural basis of the collagen-binding mode of discoidin domain receptor 2, EMBO J. 26, 4168–4176.

    Google Scholar 

  16. New England Biolabs, Inc. K. lactis Protein Expression Kit: Instruction manual. Available at http://www.neb.com/nebecomm/ManualFiles/manualE1000.pdf.

  17. Colussi, P. A., and Taron, C. H. (2005) Kluyveromyces lactis LAC4 promoter variants that lack function in bacteria but retain full function in K. lactis, Appl. Environ. Microbiol. 71, 7092–7098.

    Google Scholar 

  18. Read, J. D., Colussi, P. A., Ganatra, M. B., and Taron, C. H. (2007) Acetamide selection of Kluyveromyces lactis cells transformed with an integrative vector leads to high-frequency formation of multicopy strains. Appl. Environ. Microbiol. 73, 5088–5096.

    Google Scholar 

  19. Sugiki, T., Shimada, I., and Takahashi, H. (2008) Stable isotope labeling of protein by Kluyveromyces lactis for NMR study. J. Biomol. NMR 42, 159–162.

    Google Scholar 

  20. Invitrogen Corporation. Pichia Fermentation Process Guidelines. Available at http://toolsja.invitrogen.com/content/sfs/manuals/pichiaferm_prot.pdf.

  21. Takahashi, H., Nakanishi, T., Kami, K., Arata, Y., and Shimada, I. (2000) A novel NMR method for determining the interface of large protein-protein complexes. Nat. Struct. Biol. 7, 220–223.

    Google Scholar 

  22. Nakanishi, T., Miyazawa, M., Sakakura, M., Terasawa, H., Takahashi, H., and Shimada, I. (2002) Determination of the interface of a large protein complex by transferred cross-saturation measurements. J. Mol. Biol. 318, 245–249.

    Google Scholar 

  23. Takahashi, H., Miyazawa, M., Ina, Y., Fukunishi, Y., Mizukoshi, Y., Nakamura, H., and Shimada, I. (2006) Utilization of methyl proton resonances in cross-saturation measurement for determining the interfaces of large protein-protein complexes. J. Biomol. NMR 34, 167–177.

    Google Scholar 

  24. Shimada, I., Ueda, T., Matsumoto, M., Sakakura, M., Osawa, M., Takeuchi, K., Nishida, N., and Takahashi, H. (2008) Cross-saturation and transferred cross-saturation experiments. Prog. Nucl. Magn. Reson. Spectrosc. 54, 123–140.

    Google Scholar 

  25. Koch, V., Rüffer, H.-M., Schügerl, K., Innertsberger, E., Menzel, H., and Weis, J. (1995) Effect of antifoam agents on the medium and microbial cell properties and process performance in small and large reactors. Process Biochem. 30, 435–446.

    Google Scholar 

  26. Macauley-Patrick, S., Fazenda, M. L., McNeil, B., and Harvey, L. M. (2005) Heterologous protein production using the Pichia pastoris expression system. Yeast 22, 249–270.

    Google Scholar 

  27. Shapiro, R. I., Wen, D., Levesque, M., Hronowski, X., Gill, A., Garber, E. A., Galdes, A., Strauch, K. L., and Taylor, F. R. (2003) Expression of sonic hedgehog-Fc fusion protein in Pichia pastoris. Identification and control of post-translational, chemical, and proteolytic modifications. Protein Expr. Purif. 29, 272–283.

    Google Scholar 

  28. Files, D., Ogawa, M., Scaman, C. H., and Baldwin, S. A. (2001) A Pichia pastoris fermentation process for producing high-levels of recombinant human cystatin-C. Enzyme Microbial Technol. 29, 335–340.

    Google Scholar 

  29. Zhang, W., Hywood Potter K. J., Plantz, B. A., Schlegel, V. L., Smith, L. A., and Meagher, M. M. (2003) Pichia pastoris fermentation with mixed-feeds of glycerol and methanol: growth kinetics and production improvement. J. Ind. Microbiol. Biotechnol. 30, 210–215.

    Google Scholar 

  30. Xie, J., Zhang, L., Ye, Q., Zhou, Q., Xin, L., Du, P., and Gan, R. (2003) Angiostatin production in cultivation of recombinant Pichia pastoris fed with mixed carbon sources. Biotechnol. Lett. 25, 173–177.

    Google Scholar 

  31. McGrew, J. T., Leiske, D., Dell, B., Klinke, R., Krasts, D., Wee, S. F., Abbott, N., Armitage, R., and Harrington, K. (1997) Expression of trimeric CD40 ligand in Pichia pastoris: use of a rapid method to detect high-level expressing transformants. Gene 187, 193–200.

    Google Scholar 

  32. d’Anjou, M. C., and Daugulis, A. J. (2001) A rational approach to improving productivity in Pichia pastoris fermentation. Biotechnol. Bioeng 72, 1–11.

    Google Scholar 

  33. Cai, M., Huang, Y., Sakaguchi, K., Clore, G. M., Gronenborn, A. M., and Craigie, R. (1998) An efficient and cost-effective isotope labeling protocol for proteins expressed in Escherichia coli. J. Biomol. NMR 11, 97–102.

    Google Scholar 

  34. Zhang, Y., Liu, R., and Wu, X. (2007) The proteolytic systems and heterologous proteins degradation in the methylotrophic yeast Pichia pastoris. Annal. Microbiol. 57, 553–560.

    Google Scholar 

  35. Li, Z., Xiong, F., Lin, Q., d’Anjou, M., Daugulis, A. J., Yang, D. S., and Hew, C. L. (2001) Low-temperature increases the yield of biological active herring antifreeze protein in Pichia pastoris. Protein Expr. Purif. 21, 438–445.

    Google Scholar 

  36. Jahic, M., Gustavsson, M., Jansen, A.-K., Martinelle, M., and Enfors, S.-O. (2003) Analysis and control of proteolysis of a fusion protein in Pichia pastoris fed-batch processes. J. Biotechnol. 102, 45–53.

    Google Scholar 

  37. Jahic, M., Wallberg, F., Bollok, M., Garcia, P., and Enfors, S.-O. (2003) Temperature limited fed-batch technique for control of proteolysis in Pichia pastoris bioreactor cultures. Microbial Cell Factories 2, 6–17.

    Google Scholar 

  38. Surribas, A., Stahn, R., Montesinos, J. L., Enfors, S.-O., Valero, F., and Jahic, M. (2007) Production of Rhizopus oryzae lipase from Pichia pastoris using alternative operational strategies. J. Biotechnol. 130, 291–299.

    Google Scholar 

  39. Shi, X., Karkut T., Chamankhah, M., Alting-Mees, M., Hemmingsen, S. M., and Hegedus, D. (2003) Optimal conditions for the expression of a single-chain antibody (scFv) gene in Pichia pastoris. Protein Expr. Purif. 28, 321–330.

    Google Scholar 

  40. Yao, X. Q., Zhao, H. L., Xue, C., Zhang, W., Xiong, X. H., Wang, Z. W., Li, X. Y., and Liu, Z. M. (2009) Degradation of HSA-AX15(R13K) when expressed in Pichia pastoris can be reduced via the disruption of YPS1 gene in this yeast. J. Biotechnol. 139, 131–136.

    Google Scholar 

  41. Madduri, K., Badger, M., Li, Z.-S., Xu, X., Thornburgh, S., Evans, S., and Dhadialla, T. S. (2009) Development of stable isotope and selenomethionine labeling methods for proteins expressed in Pseudomonas fluorescens. Protein Expr. Purif. 65, 57–65.

    Google Scholar 

Download references

Acknowledgments

This work was financially supported in part by the Ministry of Economy, Trade and Industry (METI) and the New Energy and Industrial Technology Development Organization (NEDO).

Author information

Authors and Affiliations

  1. Japan Biological Informatics Consortium (JBiC), Tokyo, Japan

    Toshihiko Sugiki & Mayumi Miyazawa-Onami

  2. Biomedicinal Information Research Center (BIRC), National Institute of Advanced Industrial Science and Technology (AIST), Tokyo, Japan

    Toshihiko Sugiki, Mayumi Miyazawa-Onami, Ichio Shimada & Hideo Takahashi

  3. Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan

    Osamu Ichikawa & Ichio Shimada

  4. Department of Supramolecular Biology, Graduate School of Nanobioscience, Yokohama City University, Yokohama, Japan

    Hideo Takahashi

Authors
  1. Toshihiko Sugiki
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Osamu Ichikawa
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Mayumi Miyazawa-Onami
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Ichio Shimada
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Hideo Takahashi
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Hideo Takahashi .

Editor information

Editors and Affiliations

  1. University of Albany, State University of New York, Washington Avenue 1400, Albany, 12222, New York, USA

    Alexander Shekhtman

  2. University of Albany, State University of New York, Washington Avenue 1400, Albany, 12222, New York, USA

    David S. Burz

Rights and permissions

Reprints and permissions

Copyright information

© 2012 Springer Science+Business Media, LLC

About this protocol

Cite this protocol

Sugiki, T., Ichikawa, O., Miyazawa-Onami, M., Shimada, I., Takahashi, H. (2012). Isotopic Labeling of Heterologous Proteins in the Yeast Pichia pastoris and Kluyveromyces lactis . In: Shekhtman, A., Burz, D. (eds) Protein NMR Techniques. Methods in Molecular Biology, vol 831. Humana Press. https://doi.org/10.1007/978-1-61779-480-3_2

Download citation

  • DOI: https://doi.org/10.1007/978-1-61779-480-3_2

  • Published:

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-61779-479-7

  • Online ISBN: 978-1-61779-480-3

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation