Skip to main content
SpringerLink
Log in

High-Throughput Protein Production in Yeast

  • Protocol
  • First Online:
High-Throughput Protein Production and Purification

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2025))

Abstract

Yeasts are versatile single-celled fungi that grow to high cell densities on inexpensive media. With well-studied genetics and metabolism and a wealth of knowledge available about their propagation and growth in academic as well as industrial settings, yeasts have long been used for recombinant protein production of isolated proteins and multisubunit complexes. They can be easily adapted to high-throughput protein expression pipelines. Importantly, the outcome from small-scale expression evaluations in high-throughput mode is scalable to laboratory and industrial scales using well-established procedures. In this chapter, we offer a state-of-the-art perspective on currently available high-throughput pipelines for protein production in S. cerevisiae and P. pastoris and discuss future challenges and avenues for improvement.

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 189.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 239.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 329.99
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. Botstein D, Fink GR (2011) Yeast: an experimental organism for 21st Century biology. Genetics 189:695–704. https://doi.org/10.1534/genetics.111.130765

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Kell DB, Brown M, Davey HM et al (2005) Metabolic footprinting and systems biology: the medium is the message. Nat Rev Microbiol 3:557–565. https://doi.org/10.1038/nrmicro1177

    Article  CAS  PubMed  Google Scholar 

  3. Picotti P, Clément-Ziza M, Lam H et al (2013) A complete mass-spectrometric map of the yeast proteome applied to quantitative trait analysis. Nature 494:266–270. https://doi.org/10.1038/nature11835

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Annaluru N, Muller H, Mitchell LA et al (2014) Total synthesis of a functional designer eukaryotic chromosome. Science 344:55–58. https://doi.org/10.1126/science.1249252

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Richardson SM, Mitchell LA, Stracquadanio G et al (2017) Design of a synthetic yeast genome. Science 355:1040–1044. https://doi.org/10.1126/science.aaf4557

    Article  CAS  PubMed  Google Scholar 

  6. Marsit S, Leducq J-B, Durand É et al (2017) Evolutionary biology through the lens of budding yeast comparative genomics. Nat Rev Genet 18:581–598. https://doi.org/10.1038/nrg.2017.49

    Article  CAS  PubMed  Google Scholar 

  7. Resnick MA, Cox BS (2000) Yeast as an honorary mammal. Mutat Res 451:1–11

    Article  CAS  Google Scholar 

  8. Meehl MA, Stadheim TA (2014) Biopharmaceutical discovery and production in yeast. Curr Opin Biotechnol 30:120–127. https://doi.org/10.1016/j.copbio.2014.06.007

    Article  CAS  PubMed  Google Scholar 

  9. Mehla J, Caufield JH, Uetz P (2015) The yeast two-hybrid system: a tool for mapping protein-protein interactions. Cold Spring Harb Protoc 2015:425–430. https://doi.org/10.1101/pdb.top083345

    Article  PubMed  Google Scholar 

  10. Young KH (1998) Yeast Two-hybrid: so many interactions, (in) so little time… . Biol Reprod 58:302–311. https://doi.org/10.1095/biolreprod58.2.302

    Article  CAS  PubMed  Google Scholar 

  11. Brückner A, Polge C, Lentze N et al (2009) Yeast two-hybrid, a powerful tool for systems biology. Int J Mol Sci 10:2763–2788. https://doi.org/10.3390/ijms10062763

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Hall DA, Zhu H, Zhu X et al (2004) Regulation of gene expression by a metabolic enzyme. Science 306:482–484. https://doi.org/10.1126/science.1096773

    Article  CAS  PubMed  Google Scholar 

  13. Cereghino JL, Cregg JM (2000) Heterologous protein expression in the methylotrophic yeastPichia pastoris. FEMS Microbiol Rev 24:45–66. https://doi.org/10.1111/j.1574-6976.2000.tb00532.x

    Article  CAS  PubMed  Google Scholar 

  14. 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

    Article  PubMed  PubMed Central  Google Scholar 

  15. Gellissen G, Kunze G, Gaillardin C et al (2005) New yeast expression platforms based on methylotrophic Hansenula polymorpha and Pichia pastoris and on dimorphic Arxula adeninivorans and Yarrowia lipolytica - a comparison. FEMS Yeast Res 5:1079–1096. https://doi.org/10.1016/j.femsyr.2005.06.004

    Article  CAS  PubMed  Google Scholar 

  16. Fernández FJ, López-Estepa M, Querol-García J, Vega MC (2016) Production of protein complexes in non-methylotrophic and methylotrophic yeasts: nonmethylotrophic and methylotrophic yeasts. Adv Exp Med Biol 896:137–153. https://doi.org/10.1007/978-3-319-27216-0_9

    Article  CAS  PubMed  Google Scholar 

  17. Fernández FJ, Vega MC (2016) Choose a suitable expression host: a survey of available protein production platforms. Adv Exp Med Biol 896:15–24. https://doi.org/10.1007/978-3-319-27216-0_2

    Article  CAS  PubMed  Google Scholar 

  18. Fernández FJ, Vega MC (2013) Technologies to keep an eye on: alternative hosts for protein production in structural biology. Curr Opin Struct Biol 23:365–373. https://doi.org/10.1016/j.sbi.2013.02.002

    Article  CAS  PubMed  Google Scholar 

  19. DiDonato M, Deacon AM, Klock HE et al (2004) A scaleable and integrated crystallization pipeline applied to mining the Thermotoga maritima proteome. J Struct Funct Genom 5:133–146. https://doi.org/10.1023/B:JSFG.0000029194.04443.50

    Article  CAS  Google Scholar 

  20. Fang Z, van der Merwe RG, Warren RM et al (2015) Assessing the progress of Mycobacterium tuberculosis H37Rv structural genomics. Tuberculosis (Edinb) 95:131–136. https://doi.org/10.1016/j.tube.2014.12.005

    Article  CAS  Google Scholar 

  21. Vieth M, Sutherland JJ, Robertson DH, Campbell RM (2005) Kinomics: characterizing the therapeutically validated kinase space. Drug Discov Today 10:839–846. https://doi.org/10.1016/S1359-6446(05)03477-X

    Article  CAS  PubMed  Google Scholar 

  22. Saez NJ, Nozach H, Blemont M, Vincentelli R (2014) High throughput quantitative expression screening and purification applied to recombinant disulfide-rich venom proteins produced in E. coli. J Vis Exp 89:e51464. https://doi.org/10.3791/51464

    Article  Google Scholar 

  23. Turchetto J, Sequeira AF, Ramond L et al (2017) High-throughput expression of animal venom toxins in Escherichia coli to generate a large library of oxidized disulphide-reticulated peptides for drug discovery. Microb Cell Factories 16:6. https://doi.org/10.1186/s12934-016-0617-1

    Article  CAS  Google Scholar 

  24. Kim MD, Lee TH, Lim HK, Seo JH (2004) Production of antithrombotic hirudin in GAL1-disrupted Saccharomyces cerevisiae. Appl Microbiol Biotechnol 65:259–262. https://doi.org/10.1007/s00253-004-1598-2

    Article  CAS  PubMed  Google Scholar 

  25. Rohde JR, Trinh J, Sadowski I (2000) Multiple signals regulate GAL transcription in yeast. Mol Cell Biol 20:3880–3886

    Article  CAS  Google Scholar 

  26. Denis CL, Ferguson J, Young ET (1983) mRNA levels for the fermentative alcohol dehydrogenase of Saccharomyces cerevisiae decrease upon growth on a nonfermentable carbon source. J Biol Chem 258:1165–1171

    CAS  PubMed  Google Scholar 

  27. Gatignol A, Dassain M, Tiraby G (1990) Cloning of Saccharomyces cerevisiae promoters using a probe vector based on phleomycin resistance. Gene 91:35–41

    Article  CAS  Google Scholar 

  28. Cregg JM, Vedvick TS, Raschke WC (1993) Recent advances in the expression of foreign genes in Pichia pastoris. Biotechnology (NY) 11:905–910

    CAS  Google Scholar 

  29. Ellis SB, Brust PF, Koutz PJ et al (1985) Isolation of alcohol oxidase and two other methanol regulatable genes from the yeast Pichia pastoris. Mol Cell Biol 5:1111–1121

    Article  CAS  Google Scholar 

  30. Montoliu-Gaya L, Esquerda-Canals G, Bronsoms S, Villegas S (2017) Production of an anti-Aβ antibody fragment in Pichia pastoris and in vitro and in vivo validation of its therapeutic effect. PLoS One 12:e0181480. https://doi.org/10.1371/journal.pone.0181480

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Aw R, McKay PF, Shattock RJ, Polizzi KM (2017) Expressing anti-HIV VRC01 antibody using the murine IgG1 secretion signal in Pichia pastoris. AMB Express 7:70. https://doi.org/10.1186/s13568-017-0372-7

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Vogl T, Sturmberger L, Kickenweiz T et al (2016) A toolbox of diverse promoters related to methanol utilization: functionally verified parts for heterologous pathway expression in Pichia pastoris. ACS Synth Biol 5:172–186. https://doi.org/10.1021/acssynbio.5b00199

    Article  CAS  PubMed  Google Scholar 

  33. Purcell O, Opdensteinen P, Chen W et al (2017) Production of functional anti-ebola antibodies in Pichia pastoris. ACS Synth Biol 6(12):2183–2190. https://doi.org/10.1021/acssynbio.7b00234

    Article  CAS  PubMed  Google Scholar 

  34. Cai Y, Yao S, Zhong J et al (2017) Inhibition activity of a disulfide-stabilized diabody against basic fibroblast growth factor in lung cancer. Oncotarget 8:20187–20197. https://doi.org/10.18632/oncotarget.15556

    Article  PubMed  PubMed Central  Google Scholar 

  35. Pourasadi S, Mousavi Gargari SL, Rajabibazl M, Nazarian S (2017) Efficient production of nanobodies against urease activity ofHelicobacter pylori in Pichia pastoris. Turk J Med Sci 47:695–701. https://doi.org/10.3906/sag-1509-121

    Article  CAS  PubMed  Google Scholar 

  36. Schwarzhans J-P, Luttermann T, Wibberg D et al (2017) A mitochondrial autonomously replicating sequence from Pichia pastoris for uniform high level recombinant protein production. Front Microbiol 8:780. https://doi.org/10.3389/fmicb.2017.00780

    Article  PubMed  PubMed Central  Google Scholar 

  37. Camattari A, Goh A, Yip LY et al (2016) Characterization of a panARS-based episomal vector in the methylotrophic yeast Pichia pastoris for recombinant protein production and synthetic biology applications. Microb Cell Factories 15:139. https://doi.org/10.1186/s12934-016-0540-5

    Article  CAS  Google Scholar 

  38. Koskela EV, de Ruijter JC, Frey AD (2017) Following nature’s roadmap: folding factors from plasma cells led to improvements in antibody secretion in S. cerevisiae. Biotechnol J 12:8. https://doi.org/10.1002/biot.201600631

    Article  CAS  Google Scholar 

  39. Huang M, Bai Y, Sjostrom SL et al (2015) Microfluidic screening and whole-genome sequencing identifies mutations associated with improved protein secretion by yeast. Proc Natl Acad Sci U S A 112:E4689–E4696. https://doi.org/10.1073/pnas.1506460112

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Drew D, Newstead S, Sonoda Y et al (2008) GFP-based optimization scheme for the overexpression and purification of eukaryotic membrane proteins in Saccharomyces cerevisiae. Nat Protoc 3:784–798. https://doi.org/10.1038/nprot.2008.44

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Abatemarco J, Sarhan MF, Wagner JM et al (2017) RNA-aptamers-in-droplets (RAPID) high-throughput screening for secretory phenotypes. Nat Commun 8:332. https://doi.org/10.1038/s41467-017-00425-7

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Kota J, Gilstring CF, Ljungdahl PO (2007) Membrane chaperone Shr3 assists in folding amino acid permeases preventing precocious ERAD. J Cell Biol 176:617–628. https://doi.org/10.1083/jcb.200612100

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Henricsson C, de Jesus Ferreira MC, Hedfalk K et al (2005) Engineering of a novel Saccharomyces cerevisiae wine strain with a respiratory phenotype at high external glucose concentrations. Appl Environ Microbiol 71:6185–6192. https://doi.org/10.1128/AEM.71.10.6185-6192.2005

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Ferndahl C, Bonander N, Logez C et al (2010) Increasing cell biomass in Saccharomyces cerevisiae increases recombinant protein yield: the use of a respiratory strain as a microbial cell factory. Microb Cell Factories 9:47. https://doi.org/10.1186/1475-2859-9-47

    Article  CAS  Google Scholar 

  45. Küberl A, Schneider J, Thallinger GG et al (2011) High-quality genome sequence of Pichia pastoris CBS7435. J Biotechnol 154:312–320. https://doi.org/10.1016/j.jbiotec.2011.04.014

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

We gratefully acknowledge the support received during the preparation of this chapter. MCV has received funding from the Spanish Ministerio de Economía y Competitividad (PET2008_0101, CTQ2015-66206-C2-2-R, and SAF2015-72961-EXP), the Regional Government of Madrid (S2017/BMD-3673), and the European Commission (Framework Programme 7 (FP7)) project ComplexINC (Contract No. 279039). Abvance Biotech srl contributed with salaries (FJF). Neither funder had any role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Author information

Authors and Affiliations

  1. Abvance Biotech srl, Madrid, Spain

    Francisco J. Fernández

  2. Center for Biological Research (CIB-CSIC), Structural and Chemical Biology, Madrid, Spain

    Sara Gómez & M. Cristina Vega

Authors
  1. Francisco J. Fernández
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Sara Gómez
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. M. Cristina Vega
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Francisco J. Fernández or M. Cristina Vega .

Editor information

Editors and Affiliations

  1. Architecture et Fonction des Macromolécules Biologiques (AFMB), Unité Mixte de Recherche (UMR) 7257, Centre National de la Recherche Scientifique (CNRS), Aix-Marseille Université, Marseille cedex 9, France

    Renaud Vincentelli

Rights and permissions

Reprints and permissions

Copyright information

© 2019 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Fernández, F.J., Gómez, S., Vega, M.C. (2019). High-Throughput Protein Production in Yeast. In: Vincentelli, R. (eds) High-Throughput Protein Production and Purification. Methods in Molecular Biology, vol 2025. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9624-7_4

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-9624-7_4

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-4939-9623-0

  • Online ISBN: 978-1-4939-9624-7

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation