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Membrane Protein Production in the Yeast P. pastoris

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

Abstract

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.

Key words

  • Recombinant membrane protein
  • Yeast
  • Komagataella pastoris
  • Methanol
  • AOX1

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  • DOI: 10.1007/978-1-0716-2368-8_10
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Abbreviations

BMGY:

Buffered glycerol-complex medium

BMMY:

Buffered methanol-complex medium

BSM:

Basal salts medium

DO:

Dissolved oxygen

HPLC:

High-performance liquid chromatography

v/v:

Volume by volume

w/v:

Weight by volume

YPD:

Yeast extract peptone dextrose medium

YPG:

Yeast extract peptone glycerol medium

References

  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. https://doi.org/10.1007/978-1-61779-770-5_2

    CAS  CrossRef  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. https://doi.org/10.1186/s12934-014-0127-y

    CAS  CrossRef  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. https://doi.org/10.1126/science.1116269

    CAS  CrossRef  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. https://doi.org/10.3389/fmicb.2014.00085

    CrossRef  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. https://doi.org/10.1126/science.abc5667

    CAS  CrossRef  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. https://doi.org/10.1126/sciadv.abe5983

    CAS  CrossRef  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. https://doi.org/10.1016/j.bbamem.2020.183419

    CAS  CrossRef  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. https://doi.org/10.1016/j.pep.2007.10.013

    CAS  CrossRef  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. https://doi.org/10.1016/j.cell.2020.03.037

    CAS  CrossRef  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. https://doi.org/10.3109/09687688.2011.602219

    CAS  CrossRef  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. https://doi.org/10.1016/j.pep.2019.105524

    CAS  CrossRef  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. https://doi.org/10.1186/1475-2859-11-78

    CAS  CrossRef  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. https://doi.org/10.1016/j.ymeth.2011.08.015

    CAS  CrossRef  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. https://doi.org/10.1007/978-1-61779-770-5_10

    CAS  CrossRef  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. https://doi.org/10.1016/j.ymeth.2018.04.009

    CAS  CrossRef  PubMed  Google Scholar 

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Acknowledgments

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

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Correspondence to Roslyn M. Bill .

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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. https://doi.org/10.1007/978-1-0716-2368-8_10

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  • DOI: https://doi.org/10.1007/978-1-0716-2368-8_10

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  • Publisher Name: Humana, New York, NY

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