Skip to main content
SpringerLink
Log in

Optimising Yeast as a Host for Recombinant Protein Production (Review)

  • Protocol
  • First Online:
Recombinant Protein Production in Yeast

Part of the book series: Methods in Molecular Biology ((MIMB,volume 866))

Abstract

Having access to suitably stable, functional recombinant protein samples underpins diverse academic and industrial research efforts to understand the workings of the cell in health and disease. Synthesising a protein in recombinant host cells typically allows the isolation of the pure protein in quantities much higher than those found in the protein’s native source. Yeast is a popular host as it is a eukaryote with similar synthetic machinery to the native human source cells of many proteins of interest, while also being quick, easy, and cheap to grow and process. Even in these cells the production of some proteins can be plagued by low functional yields. We have identified molecular mechanisms and culture parameters underpinning high yields and have consolidated our findings to engineer improved yeast cell factories. In this chapter, we provide an overview of the opportunities available to improve yeast as a host system for recombinant protein production.

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. Bill RM, Henderson PJ, Iwata S, Kunji ER, Michel H, Neutze R, Newstead S, Poolman B, Tate CG, Vogel H (2011) Overcoming barriers to membrane protein structure determination. Nat Biotechnol 29:335–340

    Article  PubMed  CAS  Google Scholar 

  2. Bill RM (2001) Yeast – a panacea for the structure-function analysis of membrane proteins? Curr Genet 40:157–171

    Article  PubMed  CAS  Google Scholar 

  3. Grisshammer R, Tate CG (1995) Overexpression of integral membrane proteins for structural studies. Q Rev Biophys 28:315–422

    Article  PubMed  CAS  Google Scholar 

  4. Gelperin DM, White MA, Wilkinson ML, Kon Y, Kung LA, Wise KJ, Lopez-Hoyo N, Jiang L, Piccirillo S, Yu H, Gerstein M, Dumont ME, Phizicky EM, Snyder M, Grayhack EJ (2005) Biochemical and genetic analysis of the yeast proteome with a movable ORF collection. Genes Dev 19:2816–2826

    Article  PubMed  CAS  Google Scholar 

  5. Reilander H, Weiss HM (1998) Production of G-protein-coupled receptors in yeast. Curr Opin Biotechnol 9:510–517

    Article  PubMed  CAS  Google Scholar 

  6. Sarramegna V, Talmont F, Demange P, Milon A (2003) Heterologous expression of G-protein-coupled receptors: comparison of expression systems fron the standpoint of large-scale production and purification. Cell Mol Life Sci 60:1529–1546

    Article  PubMed  CAS  Google Scholar 

  7. Freigassner M, Pichler H, Glieder A (2009) Tuning microbial hosts for membrane protein production. Microb Cell Fact 8:69

    Article  PubMed  Google Scholar 

  8. Karlgren S, Pettersson N, Nordlander B, Mathai JC, Brodsky JL, Zeidel ML, Bill RM, Hohmann S (2005) Conditional osmotic stress in yeast: a system to study transport through aquaglyceroporins and osmostress signaling. J Biol Chem 280:7186–7193

    Article  PubMed  CAS  Google Scholar 

  9. Nyblom M, Oberg F, Lindkvist-Petersson K, Hallgren K, Findlay H, Wikstrom J, Karlsson A, Hansson O, Booth PJ, Bill RM, Neutze R, Hedfalk K (2007) Exceptional overproduction of a functional human membrane protein. Protein Expr Purif 56:110–120

    Article  PubMed  CAS  Google Scholar 

  10. Jidenko M, Nielsen RC, Sorensen TL, Moller JV, le Maire M, Nissen P, Jaxel C (2005) Crystallization of a mammalian membrane protein overexpressed in Saccharomyces cerevisiae. Proc Natl Acad Sci USA 102:11687–11691

    Article  PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  12. Tornroth-Horsefield S, Wang Y, Hedfalk K, Johanson U, Karlsson M, Tajkhorshid E, Neutze R, Kjellbom P (2006) Structural mechanism of plant aquaporin gating. Nature 439:688–694

    Article  PubMed  Google Scholar 

  13. Horsefield R, Norden K, Fellert M, Backmark A, Tornroth-Horsefield S, Terwisscha van Scheltinga AC, Kvassman J, Kjellbom P, Johanson U, Neutze R (2008) High-resolution x-ray structure of human aquaporin 5. Proc Natl Acad Sci USA 105:13327–13332

    Article  PubMed  CAS  Google Scholar 

  14. De Schutter K, Lin YC, Tiels P, Van Hecke A, Glinka S, Weber-Lehmann J, Rouze P, Van de Peer Y, Callewaert N (2009) Genome sequence of the recombinant protein production host Pichia pastoris. Nat Biotechnol 27:561–566

    Article  PubMed  Google Scholar 

  15. Goffeau A, Barrell BG, Bussey H, Davis RW, Dujon B, Feldmann H, Galibert F, Hoheisel JD, Jacq C, Johnston M, Louis EJ, Mewes HW, Murakami Y, Philippsen P, Tettelin H, Oliver SG (1996) Life with 6000 genes. Science 274:546, 563–567

    Article  PubMed  CAS  Google Scholar 

  16. Hamilton SR, Bobrowicz P, Bobrowicz B, Davidson RC, Li H, Mitchell T, Nett JH, Rausch S, Stadheim TA, Wischnewski H, Wildt S, Gerngross TU (2003) Production of complex human glycoproteins in yeast. Science 301:1244–1246

    Article  PubMed  CAS  Google Scholar 

  17. Hamilton SR, Gerngross TU (2007) Glycosylation engineering in yeast: the advent of fully humanized yeast. Curr Opin Biotechnol 18:387–392

    Article  PubMed  CAS  Google Scholar 

  18. Kjeldsen T, Ludvigsen S, Diers I, Balschmidt P, Sørensen AR, Kaarsholm NC (2002) Engineering-enhanced protein secretory expression in yeast with application to insulin. J Biol Chem 277:18245–18248

    Article  PubMed  CAS  Google Scholar 

  19. Siddiqui MAA, Perry CM (2006) Human papillomavirus quadrivalent (types 6, 11, 16, 18) recombinant vaccine (Gardasil (R)). Drugs 66:1263–1271

    Article  PubMed  Google Scholar 

  20. Miroux B, Walker JE (1996) Over-production of proteins in Escherichia coli: mutant hosts that allow synthesis of some membrane proteins and globular proteins at high levels. J Mol Biol 260:289–298

    Article  PubMed  CAS  Google Scholar 

  21. Arechaga I, Miroux B, Karrasch S, Huijbregts R, de Kruijff B, Runswick MJ, Walker JE (2000) Characterisation of new intracellular membranes in Escherichia coli accompanying large scale over-production of the b subunit of F(1)F(o) ATP synthase. FEBS Lett 482:215–219

    Article  PubMed  CAS  Google Scholar 

  22. Linares DM, Geertsma ER, Poolman B (2010) Evolved Lactococcus lactis strains for enhanced expression of recombinant membrane proteins. J Mol Biol 401:45–55

    Article  PubMed  CAS  Google Scholar 

  23. Bonander N, Hedfalk K, Larsson C, Mostad P, Chang C, Gustafsson L, Bill RM (2005) Design of improved membrane protein production experiments: quantitation of the host response. Protein Sci 14:1729–1740

    Article  PubMed  CAS  Google Scholar 

  24. Griffith DA, Delipala C, Leadsham J, Jarvis SM, Oesterhelt D (2003) A novel yeast expression system for the overproduction of quality-controlled membrane proteins. FEBS Lett 553:45–50

    Article  PubMed  CAS  Google Scholar 

  25. Bonander N, Bill RM (2009) Relieving the first bottleneck in the drug discovery pipeline: using array technologies to rationalize membrane protein production. Expert Rev Proteomics 6:501–505

    Article  PubMed  CAS  Google Scholar 

  26. Bonander N, Darby RA, Grgic L, Bora N, Wen J, Brogna S, Poyner DR, O’Neill MA, Bill RM (2009) Altering the ribosomal subunit ratio in yeast maximizes recombinant protein yield. Microb Cell Fact 8:10

    Article  PubMed  Google Scholar 

  27. Bawa Z, Bland CE, Bonander N, Bora N, Cartwright SP, Clare M, Conner MT, Darby RA, Dilworth MV, Holmes WJ, Jamshad M, Routledge SJ, Gross SR, Bill RM (2011) Understanding the yeast host cell response to recombinant membrane protein production. Biochem Soc Trans 39:1113

    CAS  Google Scholar 

  28. Santiago TC, Mamoun CB (2003) Genome expression analysis in yeast reveals novel transcriptional regulation by inositol and choline and new regulatory functions for Opi1p, Ino2p and Ino4p. J Biol Chem 278:38723–38730

    Article  PubMed  CAS  Google Scholar 

  29. Jiranek V, Graves JA, Henry SA (1998) Pleiotropic effects of the opi1 regulatory mutation of yeast: its effects on growth and on phospholipid and inositol metabolism. Microbiology 144:2739–2748

    Article  PubMed  CAS  Google Scholar 

  30. Sohn SB, Graf AB, Kim TY, Gasser B, Maurer M, Ferrer P, Mattanovich D, Lee SY (2010) Genome-scale metabolic model of methylotrophic yeast Pichia pastoris and its use for in silico analysis of heterologous protein production. Biotechnol J 5:705–715

    Article  PubMed  CAS  Google Scholar 

  31. De Alwis DM, Dutton RL, Scharer J, Moo-Young M (2007) Statistical methods in media optimization for batch and fed-batch animal cell culture. Bioprocess Biosyst Eng 30:107–113

    Article  PubMed  CAS  Google Scholar 

  32. Stiens LR, Buntemeyer H, Lutkemeyer D, Lehmann J, Bergmann A, Weglohner W (2000) Development of serum-free bioreactor production of recombinant human thyroid stimulating hormone receptor. Biotechnol Prog 16:703–709

    Article  PubMed  CAS  Google Scholar 

  33. van der Valk J, Brunner D, De Smet K, Fex Svenningsen A, Honegger P, Knudsen LE, Lindl T, Noraberg J, Price A, Scarino ML, Gstraunthaler G (2010) Optimization of chemically defined cell culture media – replacing fetal bovine serum in mammalian in vitro methods. Toxicol In Vitro 24:1053–1063

    Article  PubMed  Google Scholar 

  34. Andre N, Cherouati N, Prual C, Steffan T, Zeder-Lutz G, Magnin T, Pattus F, Michel H, Wagner R, Reinhart C (2006) Enhancing functional production of G protein-coupled receptors in Pichia pastoris to levels required for structural studies via a single expression screen. Protein Sci 15:1115–1126

    Article  PubMed  CAS  Google Scholar 

  35. Bonander N, Jamshad M, Hu K, Farquhar MJ, Stamataki Z, Balfe P, McKeating JA, Bill RM (2011) Structural characterization of CD81-Claudin-1 hepatitis C virus receptor complexes. Biochem Soc Trans 39:537–540

    Article  PubMed  CAS  Google Scholar 

  36. Holmes WJ, Darby RA, Wilks MD, Smith R, Bill RM (2009) Developing a scalable model of recombinant protein yield from Pichia pastoris: the influence of culture conditions, biomass and induction regime. Microb Cell Fact 8:35

    Article  PubMed  Google Scholar 

  37. Verduyn C, Postma E, Scheffers WA, Van Dijken JP (1992) Effect of benzoic acid on metabolic fluxes in yeasts: a continuous-culture study on the regulation of respiration and alcoholic fermentation. Yeast 8:501–517

    Article  PubMed  CAS  Google Scholar 

  38. Abelovska L, Bujdos M, Kubova J, Petrezselyova S, Nosek J, Tomaska L (2007) Comparison of element levels in minimal and complex yeast media. Can J Microbiol 53:533–535

    Article  PubMed  CAS  Google Scholar 

  39. Burkholder PR, McVeigh I, Moyer D (1944) Studies on some growth factors of yeasts. J Bacteriol 48:385–391

    PubMed  CAS  Google Scholar 

  40. Wickerham LJ (1946) A critical evaluation of the nitrogen assimilation tests commonly used in the classification of yeasts. J Bacteriol 52:293–301

    CAS  Google Scholar 

  41. Wickerman LJ (1951) Taxonomy of yeasts. US Dept Agri Tech Bull 1029:1–56

    Google Scholar 

  42. Henricsson C, de Jesus Ferreira MC, Hedfalk K, Elbing K, Larsson C, Bill RM, Norbeck J, Hohmann S, Gustafsson L (2005) Engineering of a novel Saccharomyces cerevisiae wine strain with a respiratory phenotype at high external glucose concentrations. Appl Environ Microbiol 71:6185–6192

    Article  PubMed  CAS  Google Scholar 

  43. Ferndahl C, Bonander N, Logez C, Wagner R, Gustafsson L, Larsson C, Hedfalk K, Darby RA, Bill RM (2010) Increasing cell biomass in Saccharomyces cerevisiae increases recombinant protein yield: the use of a respiratory strain as a microbial cell factory. Microb Cell Fact 9:47

    Article  PubMed  Google Scholar 

  44. van de Laar T, Visser C, Holster M, Lopez CG, Kreuning D, Sierkstra L, Lindner N, Verrips T (2007) Increased heterologous protein production by Saccharomyces cerevisiae growing on ethanol as sole carbon source. Biotechnol Bioeng 96:483–494

    Article  PubMed  Google Scholar 

  45. Gaspar ML, Aregullin MA, Jesch SA, Henry SA (2006) Inositol induces a profound alteration in the pattern and rate of synthesis and turnover of membrane lipids in Saccharomyces cerevisiae. J Biol Chem 281:22773–22785

    Article  PubMed  CAS  Google Scholar 

  46. Weuster-Botz D (2000) Experimental design for fermentation media development: statistical design or global random search? J Biosci Bioeng 90:473–483

    PubMed  CAS  Google Scholar 

  47. Rezessy-Szabo JM, Nguyen QD, Hoschke A (2000) Optimisation of composition of media for the production of amylolytic enzymes by Thermomyces lanuginosus ATCC 34626. Food Technol Biotechnol 38:229–234

    Google Scholar 

  48. Ratnam BVV, Subba Rao S, Mendu DR, Narasimha Rao M, Ayyanna C (2005) Optimization of medium constituents and fermentation conditions for the production of ethanol from palmyra jaggery using response surface methodology. World J Microbiol Biotechnol 21:399–404

    Article  CAS  Google Scholar 

  49. Olson BH, Johnson MJ (1949) Factors producing high yeast yields in synthetic media. J Bacteriol 57:235–246

    CAS  Google Scholar 

  50. Gasser B, Maurer M, Gach J, Kunert R, Mattanovich D (2006) Engineering of Pichia pastoris for improved production of antibody fragments. Biotechnol Bioeng 94:353–361

    Article  PubMed  CAS  Google Scholar 

  51. Travers KJ, Patil CK, Wodicka L, Lockhart DJ, Weissman JS, Walter P (2000) Functional and genomic analyses reveal an essential coordination between the unfolded protein response and ER-associated degradation. Cell 101:249–258

    Article  PubMed  CAS  Google Scholar 

  52. Perlman D, O’Brien E (1954) Characteristics of a cobalt tolerant culture of Saccharomyces cerevisiae. J Bacteriol 68:167–170

    PubMed  CAS  Google Scholar 

  53. Bennett A, Rowe RI, Soch N, Eckhert CD (1999) Boron stimulates yeast (Saccharomyces cerevisiae) growth. J Nutr 129:2236–2238

    PubMed  CAS  Google Scholar 

Download references

Acknowledgements

This work was supported by the European Commission via contract LSHG-CT-2004-504601 (E-MeP), LSHG-CT-2006-037793 (OptiCryst) and Grant 201924 (EDICT) to RMB. The BBSRC supports bioreactors and a flow microcalorimeter in the RMB Laboratory through an REI award.

Author information

Authors and Affiliations

  1. Department of Chemical and Biological Engineering, Chalmers University of Technology, Gothenburg, Sweden

    Nicklas Bonander

  2. School of Life & Health Sciences and Aston Research Centre for Healthy Ageing, Aston University, Birmingham, UK

    Roslyn M. Bill

Authors
  1. Nicklas Bonander
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Roslyn M. Bill
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Roslyn M. Bill .

Editor information

Editors and Affiliations

  1. School of Life and Health Sciences, Aston University, Aston Triangle, Birmingham, B4 7ET, United Kingdom

    Roslyn M. Bill

Rights and permissions

Reprints and permissions

Copyright information

© 2012 Springer Science+business Media, LLC

About this protocol

Cite this protocol

Bonander, N., Bill, R.M. (2012). Optimising Yeast as a Host for Recombinant Protein Production (Review). In: Bill, R. (eds) Recombinant Protein Production in Yeast. Methods in Molecular Biology, vol 866. Humana Press. https://doi.org/10.1007/978-1-61779-770-5_1

Download citation

  • DOI: https://doi.org/10.1007/978-1-61779-770-5_1

  • Published:

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-61779-769-9

  • Online ISBN: 978-1-61779-770-5

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation