, Volume 11, Issue 2, pp 403–413 | Cite as

Pichia pastoris is a valuable host for the expression of genes encoding membrane proteins from the hyperthermophilic Archeon Pyrococcus abyssi

  • Cécile Labarre
  • Herman van Tilbeurgh
  • Karine Blondeau
Method Paper


We present here the experimental strategies, first results and identified bottlenecks of a structural genomics initiative on membrane proteins of the hyperthermophilic archaea Pyrococcus abyssi. Five ORFs coding for putative membrane proteins have been cloned and expressed in the methylotrophic Pichia pastoris expression system, using two different constructs, with or without the signal sequence α-mating factor of Saccharomyces cerevisiae. A c-myc epitope and 6 His codons were added at the 3′-end of the targeted genes to allow immunodetection of the recombinant proteins and to facilitate their further purification. We have selected at least one producer clone for each protein of interest and for almost every construction. All the membrane proteins were produced in Erlenmeyer flasks culture and in fed-batch cultivation for large-scale preparation. The proteins were detected in the membrane fractions of P. pastoris. Production efficiencies were relatively low in both production conditions but the quantities of biomass obtained during fed-batch cultivation have allowed us to collect sufficient amount of material for further purification. The proteins were extracted, solubilized and partially purified. Large-scale purification will be necessary for further structural work.


Pichia pastoris Structural genomics Membrane protein Archaea Pyrococcus abyssi 


  1. Adams MWW (1999) The biochemical diversity of life near and above 100 degrees C in marine environments. J Appl Microbiol 85:108S–117SGoogle Scholar
  2. Bendtsen JD, Nielsen H, von Heijne G, Brunak S (2004) Improved prediction of signal peptides: signalP 3.0. J Mol Biol 340:783–795PubMedCrossRefGoogle Scholar
  3. Cai J, Daoud R, Deorges E, Gros P (2001) Functional expression of multidrug resistance protein 1 in P. pastoris. Biochemistry 40:8307–8316PubMedCrossRefGoogle Scholar
  4. Cereghino JL, Cregg JM (2000) Heterologous protein expression in the methylotrophic yeast Pichia pastoris. FEMS Microbiol Rev 24:45–66PubMedCrossRefGoogle Scholar
  5. Cereghino GP, Cereghino JL, Ilgen C, Cregg JM (2002) Production of recombinant proteins in fermenter cultures of the yeast Pichia pastoris. Curr Opin Biotechnol 13:329–32PubMedCrossRefGoogle Scholar
  6. Cohen GN, Barbe V, Flament D, Galperin M, Heilig R, Lecompte O, Poch O, Prieur D, Quérellou J, Ripp R, Thierry JC, Van der Oost J, Weissenbach J, Zivanovic Y, Forterre P (2003) An integrated analysis of the genome of the hyperthermophilic archaeon Pyrococcus abyssi. Mol Microbiol 47:1495–1512PubMedCrossRefGoogle Scholar
  7. Cohen E, Goldshleger R, Shainskaya A, Tal DM, Ebel C, le Maire M, Karlish SJ (2005) Purification of Na+, K+-ATPase expressed in Pichia pastoris reveals an essential role of phospholipid-protein interactions. J Biol Chem 280:16610–16618PubMedCrossRefGoogle Scholar
  8. De Jong LA, Grunewald S, Franke JP, Uges DR, Bischoff R (2004) Purification and characterization of the recombinant human dopamine D2S receptor from Pichia pastoris. Protein Expr Purif 33:176–184PubMedCrossRefGoogle Scholar
  9. Erauso G, Reysenbach AL, Godfroy A, Meunier JR, Crump B, Partensky F, Baross JA, Marteinsson V, Barbier G, Pace NR, Prieur D (1993) Pyrococcus abyssi sp. nov., a new hyperthermophilic archaeon isolated from a deep-sea hydrothermal vent. Arch Microbiol 160:338–349CrossRefGoogle Scholar
  10. Forterre P, Filee J, Myllykallio H (2003) Origin and evolution of DNA and DNA replication machineries. In: The Genetic Code and the Origin of the Life, L. Ribas, ed. Landes BioscienceGoogle Scholar
  11. Forterre P, Gribaldo S, Brochier C (2005) Luca: the last universal common ancestor. Med Sci 21:860–865Google Scholar
  12. Higgins DR, Cregg JM (1998) Introduction to Pichia pastoris. Methods Mol Biol 103:1–15PubMedGoogle Scholar
  13. Hofmann K, Stoffel W (1993) Tmbase—a database of membrane spanning proteins segments. Biol Chem Hoppe-Seyler 374:166Google Scholar
  14. Hollenberg CP, Gellissen G (1997) Production of recombinant proteins by methylotrophic yeasts. Curr Opin Biotechnol 8:554–560PubMedCrossRefGoogle Scholar
  15. Karlsson M, Fotiadis D, Sjovall S, Johansson I, Hedfalk K, Engel A, Kjellbom P. (2003) Reconstitution of water channel function of an aquaporin overexpressed and purified from Pichia pastoris. FEBS Lett 537:68–72PubMedCrossRefGoogle Scholar
  16. Laborde C, Chemardin P, Bigey F, Combarnous Y, Moulin G, Boze H. (2004) Overexpression of ovine leptin in Pichia pastoris: physiological yeast response to leptin production and characterization of the recombinant hormone. Yeast 21:249–63PubMedCrossRefGoogle Scholar
  17. Laemmli UK (1970) Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature 227:680–685PubMedCrossRefGoogle Scholar
  18. Lesley SA, Kuhn P, Godzik A, Deacon AM, Mathews I, Kreusch A, Spraggon G, Klock HE, McMullan D, Shin T, Vincent J, Robb A, Brinen LS, Miller MD, McPhillips TM, Miller MA, Scheibe D, Canaves JM, Guda C, Jaroszewski L, Selby TL, Elsliger MA, Wooley J, Taylor SS, Hodgson KO, Wilson IA, Schultz PG, Stevens RC (2002) Structural genomics of the Thermotoga maritima proteome implemented in a high-throughput structure determination pipeline. Proc Natl Acad Sci USA 99:11664–11669PubMedCrossRefGoogle Scholar
  19. Macauley-Patrick S, Fazenda ML, McNeil B, Harvey LM (2005) Heterologous protein production using the Pichia pastoris expression system. Yeast 22:249–270PubMedCrossRefGoogle Scholar
  20. Makino S, Suzuki M, Zivanovic Y, Myllykallio H, Forterre P (2001) Bacterial genomic reorganization upon DNA replication. Science 292:803aCrossRefGoogle Scholar
  21. Morsomme P, Chami M, Marco S, Nader J, Ketchum KA, Goffeau A, Rigaud JL (2002) Characterization of a hyperthermophilic P-type ATPase from Methanococcus jannaschii expressed in yeast. J Biol Chem 277:29608–29616PubMedCrossRefGoogle Scholar
  22. Myllykallio H, Lopez P, Lopez-Garcia P, Heilig R, Saurin W, Zivanovic Y, Philippe H., Forterre P (2000) Bacterial mode of replication with eukaryotic-like machinery in a hyperthermophilic archaeon. Science 288:2212–2215PubMedCrossRefGoogle Scholar
  23. Opekarova M, Tanner W (2003) Specific lipid requirements of membrane proteins—a putative bottleneck in heterologous expression. Biochim Biophys Acta 1610:11–22PubMedCrossRefGoogle Scholar
  24. Quevillon-Cheruel S, Collinet B, Zhou CZ, Minard P, Blondeau K, Henkes G, Aufrere R, Coutant J, Guittet E, Lewit-Bentley A, Leulliot N, Ascone I, Sorel I, Savarin P, de La Sierra Gallay IL, de la Torre F, Poupon A, Fourme R, Janin J, van Tilbeurgh H (2003) A structural genomics initiative on yeast proteins. J Synchrotron Radiat 10:4–8PubMedCrossRefGoogle Scholar
  25. Sambrook J, Russel D W (2001) Molecular cloning: a laboratory manual. 3rd edn, Cold Spring Harbor Laboratory Press, New YorkGoogle Scholar
  26. Sarramegna V, Demange P, Milon A, Talmont F (2002a) Optimizing functional versus total expression of the human μ-opioid receptor in Pichia pastoris. Protein Expr Purif 24:212–220CrossRefGoogle Scholar
  27. Sarramegna V, Talmont F, de Seree Roch M, Milon A, Demange P (2002b) Green fluorescent protein as a reporter of human mu-opioid receptor overexpression and localization in the methylotrophic yeast Pichia pastoris. J Biotechnol 9:23–39CrossRefGoogle Scholar
  28. Sarramegna V, Talmont F, Demange P, Milon A (2003) Heterologous expression of G-protein-coupled receptors:comparison of expression systems from the standpoint of large-scale production and purification. Cell Mol Life Sci 60:1529–1546PubMedCrossRefGoogle Scholar
  29. Shrestha B, Blondeau K, Stevens WF, Hegarat FL (2004) Expression of chitin deacetylase from Colletotrichum lindemuthianum in Pichia pastoris: purification and characterization. Protein Expr Purif 38:196–204PubMedCrossRefGoogle Scholar
  30. Talmont F, Sidobre S, Demange P, Milon A, Emorine LJ. (1996) Expression and pharmacological characterization of the human mu-opioid receptor in the methylotrophic yeast Pichia pastoris. FEBS Lett 394:268–272PubMedCrossRefGoogle Scholar
  31. Weiss HM, Grisshammer R (2002) Purification and characterization of the human adenosine A2a receptor functionally expressed in Escherichia coli. Eur J Biochem 269:82–92PubMedCrossRefGoogle Scholar
  32. Weiss HM, Haase W, Michel H, Reilfinder H (1995) Expression of functional mouse 5-HTsA serotonin receptor in the methylotrophic yeast Pichia pastoris: pharmacological characterizationand localization. FEBS Lett 377:451–456PubMedCrossRefGoogle Scholar
  33. Weiss HM, Haase W, Michel H, Reilander H (1998) Comparative biochemical and pharmacological characterization of the mouse 5HT5A 5-hydroxytryptamine receptor and the human beta2-adrenergic receptor produced in the methylotrophic yeast Pichia pastoris. Biochem J 330:1137–1147PubMedGoogle Scholar
  34. Werten PJ, Hasler L, Koenderink JB, Klaassen CH, de Grip WJ, Engel A, Deen PM (2001) Large-scale purification of functional recombinant human aquaporin-2. FEBS Lett 504:200–205PubMedCrossRefGoogle Scholar

Copyright information

© Springer 2006

Authors and Affiliations

  • Cécile Labarre
    • 1
  • Herman van Tilbeurgh
    • 2
  • Karine Blondeau
    • 2
  1. 1.Institut de Génétique et Microbiologie (UMR 8621 CNRS Université Paris XI)Université Paris-SudOrsayFrance
  2. 2.Institut de Biochimie et de Biophysique Moléculaire et Cellulaire (UMR 8619 CNRS Université Paris XI)Université Paris-SudOrsayFrance

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