Expression Screening of Integral Membrane Proteins by Fusion to Fluorescent Reporters

  • Louise E. Bird
  • Joanne E. Nettleship
  • Valtteri Järvinen
  • Heather Rada
  • Anil Verma
  • Raymond J. Owens
Part of the Advances in Experimental Medicine and Biology book series (AEMB, volume 922)

Abstract

The production of recombinant integral membrane proteins for structural and functional studies remains technically challenging due to their relatively low levels of expression. To address this problem, screening strategies have been developed to identify the optimal membrane sequence and expression host for protein production. A common approach is to genetically fuse the membrane protein to a fluorescent reporter, typically Green Fluorescent Protein (GFP) enabling expression levels, localization and detergent solubilisation to be assessed. Initially developed for screening the heterologous expression of bacterial membrane proteins in Escherichia coli, the method has been extended to eukaryotic hosts, including insect and mammalian cells. Overall, GFP-based expression screening has made a major impact on the number of membrane protein structures that have been determined in the last few years.

Keywords

Integral membrane protein Green fluorescent protein Insect cells Escherichia coli Saccharomyces cerevisiae Pichia pastoris HEK 293 cells 

Notes

Acknowledgments

The OPPF-UK is funded by the Medical Research Council, UK (grant MR/K018779/1).

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Copyright information

© Springer International Publishing Switzerland 2016

Authors and Affiliations

  • Louise E. Bird
    • 1
    • 2
  • Joanne E. Nettleship
    • 1
    • 2
  • Valtteri Järvinen
    • 1
    • 2
  • Heather Rada
    • 1
    • 2
  • Anil Verma
    • 1
    • 2
  • Raymond J. Owens
    • 1
    • 2
  1. 1.OPPF-UK, The Research Complex at Harwell, Rutherford Appleton Laboratory HarwellOxfordUK
  2. 2.Division of Structural Biology, Henry Wellcome Building for Genomic MedicineUniversity of OxfordOxfordUK

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