Extremophiles

, 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

Abstract

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.

Keywords

Pichia pastoris Structural genomics Membrane protein Archaea Pyrococcus abyssi 

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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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