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Screening for High-Yielding Saccharomyces cerevisiae Clones: Using a Green Fluorescent Protein Fusion Strategy in the Production of Membrane Proteins

  • David Drew
  • Hyun KimEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 866)

Abstract

The overproduction of eukaryotic membrane proteins in milligram quantities is a major bottleneck for their further biochemical and structural investigation. Production trials exploring a range of input factors can be rationalized to improve the likelihood of success. Here we discuss some of these factors in combination with the use of a GFP-based Saccharomyces cerevisiae system that enables a quick turnaround time from clone construction to production trials. Since membrane-integrated levels do not necessarily correlate with the amount of functional recombinant protein, we also include the use of fluorescence-detection size exclusion chromatography (FSEC). Using FSEC, the quality of the recombinant material can also be rapidly evaluated as demonstrated for the functional production of the rat vesicular glutamate transporter (VGLUT2) and the human glucose transporter (GLUT1) (5).

Key words

Membrane protein Overproduction S. cerevisiae Fluorescence-detection size exclusion chromatography 

Notes

Acknowledgments

This work was supported by the Royal Society (United Kingdom) through a University Research Fellowship to DD and by a Basic Science Research Program grant through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (NRF0409-20100093) to HK.

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Copyright information

© Springer Science+business Media, LLC 2012

Authors and Affiliations

  1. 1.Division of Molecular BiosciencesMembrane Protein Crystallography Group, Imperial CollegeLondonUK
  2. 2.Laboratory of Membrane Biology, School of Biological SciencesSeoul National UniversitySeoulSouth Korea

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