Screening for High-Yielding Pichia pastoris Clones: The Production of G Protein-Coupled Receptors as a Case Study

  • Shweta Singh
  • Adrien Gras
  • Cedric Fiez-Vandal
  • Magdalena Martinez
  • Renaud Wagner
  • Bernadette Byrne
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 866)

Abstract

Pichia pastoris is an established host for the production of a wide range of recombinant proteins including membrane proteins. The system has a particularly good track record for the production of G protein-coupled receptors (GPCRs). Generation and screening of expression clones with this system use standard molecular biology techniques. Multiple clones can be generated and screened in a matter of a few weeks making this similar to Escherichia coli in terms of speed. In addition, basic buffer components and the lack of expensive equipment make small-scale expression screening in P. pastoris very cost-effective. Here we describe the procedures used for small-scale GPCR production screening.

Key words

Small-scale expression Western blot analysis Functional analysis Optimization trials 

Notes

Acknowledgments

This research was funded by the MepNet consortium, BBSRC, and GlaxoSmithKline.

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Copyright information

© Springer Science+business Media, LLC 2012

Authors and Affiliations

  • Shweta Singh
    • 1
  • Adrien Gras
    • 1
  • Cedric Fiez-Vandal
    • 2
  • Magdalena Martinez
    • 1
  • Renaud Wagner
    • 2
  • Bernadette Byrne
    • 1
  1. 1.Division of Molecular BiosciencesImperial College LondonLondonUK
  2. 2.Département Récepteurs et Protéines MembranairesCentre National de la Recherche Scientifique, Ecole Supérieure de Biotechnologie de StrasbourgIllkirchFrance

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