Skip to main content

Membrane Protein Production in the Yeast P. pastoris

  • Protocol
  • First Online:
Heterologous Expression of Membrane Proteins

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2507))


The first crystal structures of recombinant mammalian membrane proteins were solved using high-quality protein that had been produced in yeast cells. One of these, the rat Kv1.2 voltage-gated potassium channel, was synthesized in Pichia pastoris. Since then, this yeast species has remained a consistently popular choice of host for synthesizing eukaryotic membrane proteins because it is quick, easy, and cheap to culture and is capable of posttranslational modification. Very recent structures of recombinant membrane proteins produced in P. pastoris include a series of X-ray crystallography structures of the human vitamin K epoxide reductase and a cryo-electron microscopy structure of the TMEM206 proton-activated chloride channel from pufferfish. P. pastoris has also been used to structurally and functionally characterize a range of membrane proteins including tetraspanins, aquaporins, and G protein-coupled receptors. This chapter provides an overview of the methodological approaches underpinning these successes.

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

Access this chapter

USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
USD 129.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 169.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 249.99
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



Buffered glycerol-complex medium


Buffered methanol-complex medium


Basal salts medium


Dissolved oxygen


High-performance liquid chromatography


Volume by volume


Weight by volume


Yeast extract peptone dextrose medium


Yeast extract peptone glycerol medium


  1. Darby RA, Cartwright SP, Dilworth MV, Bill RM (2012) Which yeast species shall I choose? Saccharomyces cerevisiae versus Pichia pastoris (review). Methods Mol Biol 866:11–23.

    Article  CAS  PubMed  Google Scholar 

  2. Bawa Z, Routledge SJ, Jamshad M, Clare M, Sarkar D, Dickerson I, Ganzlin M, Poyner DR, Bill RM (2014) Functional recombinant protein is present in the pre-induction phases of Pichia pastoris cultures when grown in bioreactors, but not shake-flasks. Microb Cell Factories 13(1):127.

    Article  CAS  Google Scholar 

  3. Long SB, Campbell EB, Mackinnon R (2005) Crystal structure of a mammalian voltage-dependent Shaker family K+ channel. Science 309(5736):897–903.

    Article  CAS  PubMed  Google Scholar 

  4. Bill RM (2014) Playing catch-up with Escherichia coli: using yeast to increase success rates in recombinant protein production experiments. Front Microbiol 5:85.

    Article  PubMed  PubMed Central  Google Scholar 

  5. Liu S, Li S, Shen G, Sukumar N, Krezel AM, Li W (2021) Structural basis of antagonizing the vitamin K catalytic cycle for anticoagulation. Science 371(6524):eabc5667.

    Article  CAS  PubMed  Google Scholar 

  6. Deng Z, Zhao Y, Feng J, Zhang J, Zhao H, Rau MJ, Fitzpatrick JAJ, Hu H, Yuan P (2021) Cryo-EM structure of a proton-activated chloride channel TMEM206. Sci Adv 7(9):eabe5983.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Ayub H, Clare M, Milic I, Chmel NP, Boning H, Devitt A, Krey T, Bill RM, Rothnie AJ (2020) CD81 extracted in SMALP nanodiscs comprises two distinct protein populations within a lipid environment enriched with negatively charged headgroups. Biochim Biophys Acta Biomembr 1862(11):183419.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Jamshad M, Rajesh S, Stamataki Z, McKeating JA, Dafforn T, Overduin M, Bill RM (2008) Structural characterization of recombinant human CD81 produced in Pichia pastoris. Protein Expr Purif 57(2):206–216.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Kitchen P, Salman MM, Halsey AM, Clarke-Bland C, MacDonald JA, Ishida H, Vogel HJ, Almutiri S, Logan A, Kreida S, Al-Jubair T, Winkel Missel J, Gourdon P, Tornroth-Horsefield S, Conner MT, Ahmed Z, Conner AC, Bill RM (2020) Targeting aquaporin-4 subcellular localization to treat central nervous system edema. Cell 181(4):784–799.e719.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Oberg F, Sjohamn J, Conner MT, Bill RM, Hedfalk K (2011) Improving recombinant eukaryotic membrane protein yields in Pichia pastoris: the importance of codon optimization and clone selection. Mol Membr Biol 28(6):398–411.

    Article  CAS  PubMed  Google Scholar 

  11. Wiseman DN, Otchere A, Patel JH, Uddin R, Pollock NL, Routledge SJ, Rothnie AJ, Slack C, Poyner DR, Bill RM, Goddard AD (2020) Expression and purification of recombinant G protein-coupled receptors: a review. Protein Expr Purif 167:105524.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Shiroishi M, Tsujimoto H, Makyio H, Asada H, Yurugi-Kobayashi T, Shimamura T, Murata T, Nomura N, Haga T, Iwata S, Kobayashi T (2012) Platform for the rapid construction and evaluation of GPCRs for crystallography in Saccharomyces cerevisiae. Microb Cell Factories 11:78.

    Article  CAS  Google Scholar 

  13. Shiroishi M, Kobayashi T, Ogasawara S, Tsujimoto H, Ikeda-Suno C, Iwata S, Shimamura T (2011) Production of the stable human histamine H(1) receptor in Pichia pastoris for structural determination. Methods 55(4):281–286.

    Article  CAS  PubMed  Google Scholar 

  14. Routledge SJ, Clare M (2012) Setting up a bioreactor for recombinant protein production in yeast. Methods Mol Biol 866:99–113.

    Article  CAS  PubMed  Google Scholar 

  15. Dilworth MV, Piel MS, Bettaney KE, Ma P, Luo J, Sharples D, Poyner DR, Gross SR, Moncoq K, Henderson PJF, Miroux B, Bill RM (2018) Microbial expression systems for membrane proteins. Methods 147:3–39.

    Article  CAS  PubMed  Google Scholar 

Download references


We acknowledge funding from the ERACoBioTech MeMBrane project and BBSRC (BB/R02152X/1) to A.D.G., A.J.R., and R.M.B.

Author information

Authors and Affiliations


Corresponding author

Correspondence to Roslyn M. Bill .

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2022 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Ayub, H. et al. (2022). Membrane Protein Production in the Yeast P. pastoris. In: Mus-Veteau, I. (eds) Heterologous Expression of Membrane Proteins. Methods in Molecular Biology, vol 2507. Humana, New York, NY.

Download citation

  • DOI:

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2367-1

  • Online ISBN: 978-1-0716-2368-8

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics