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GFP-based optimization scheme for the overexpression and purification of eukaryotic membrane proteins in Saccharomyces cerevisiae

Nature Protocols volume 3, pages 784–798 (2008)Cite this article

Abstract

It is often difficult to produce eukaryotic membrane proteins in large quantities, which is a major obstacle for analyzing their biochemical and structural features. To date, yeast has been the most successful heterologous overexpression system in producing eukaryotic membrane proteins for high-resolution structural studies. For this reason, we have developed a protocol for rapidly screening and purifying eukaryotic membrane proteins in the yeast Saccharomyces cerevisiae. Using this protocol, in 1 week many genes can be rapidly cloned by homologous recombination into a 2 μ GFP-fusion vector and their overexpression potential determined using whole-cell and in-gel fluorescence. The quality of the overproduced eukaryotic membrane protein-GFP fusions can then be evaluated over several days using confocal microscopy and fluorescence size-exclusion chromatography (FSEC). This protocol also details the purification of targets that pass our quality criteria, and can be scaled up for a large number of eukaryotic membrane proteins in either an academic, structural genomics or commercial environment.

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Figure 1
Figure 2: Cloning by homologous recombination into 2 μ Saccharomyces cerevisiae GFP-fusion vector.
Figure 3: Estimating reliable expression levels of eukaryotic membrane protein-GFP fusions from S. cerevisiae whole-cell fluorescent measurements.
Figure 4: Examples of membrane protein GFP-fusion localization in S. cerevisiae as monitored by confocal microscopy.
Figure 5: Determining the monodispersity of membrane protein-GFP fusions in different detergents using fluorescence size-exclusion chromatography (FSEC).
Figure 6: Flowchart illustrating the purification of eukaryotic membrane proteins from GFP-fusions.
Figure 7: Examples of purified eukaryotic transport proteins.

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Acknowledgements

We thank Jan-Willem de Gier, Mitsunori Shiroishi, Shuichiro Goda and the reviewers for critically reading the manuscript and for useful comments. D.D. was a recipient of an European Molecular Biology Organization (EMBO) long-term fellowship. Funded by grants from the European Membrane Protein Consortium (E-MEP), the Membrane Protein Structure Initiative (MPSI) and the Biotechnology and Biological Sciences Research Council (BBSRC) (to S.I.). The project was also supported by grant number R01GM081827 from the National Institute of General Medical Sciences (to G.v.H. and H.K.); the content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute of General Medical Sciences or the National Institutes of Health.

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Authors and Affiliations

  1. Division of Molecular Biosciences, Department of Life Sciences, Membrane Protein Crystallography Group, Imperial College of London, London, SW7 2AZ, UK

    David Drew, Simon Newstead, Yo Sonoda & So Iwata

  2. Drug Discovery Research Department, Central Research Laboratories, Kaken Pharmaceutical Co., Ltd, 14 Shinomiya, Minamikawara-cho, Yamashina-ku, Kyoto-shi, Kyoto, 607-8042, Japan

    Yo Sonoda

  3. Department of Biochemistry and Biophysics, Stockholm Center for Biomembrane Research, Stockholm University, Stockholm, SE-106 91, Sweden

    Hyun Kim & Gunnar von Heijne

  4. Japan Science and Technology Agency, ERATO, Human Crystallography Project, Yoshida Konoe, Sakyo-ku, Kyoto, 606-851, Japan

    So Iwata

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  1. David Drew
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Correspondence to David Drew or So Iwata.

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Drew, D., Newstead, S., Sonoda, Y. et al. GFP-based optimization scheme for the overexpression and purification of eukaryotic membrane proteins in Saccharomyces cerevisiae. Nat Protoc 3, 784–798 (2008). https://doi.org/10.1038/nprot.2008.44

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