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    Proc Natl Acad Sci U S A. 2007 Aug 28;104(35):13936-41. Epub 2007 Aug 20.

    High-throughput fluorescent-based optimization of eukaryotic membrane protein overexpression and purification in Saccharomyces cerevisiae.

    Newstead S, Kim H, von Heijne G, Iwata S, Drew D.

    Source

    Division of Molecular Biosciences, Membrane Protein Crystallography Group, Imperial College, London SW7 2AZ, United Kingdom.

    Abstract

    Eukaryotic membrane proteins are often difficult to produce in large quantities, which is a significant obstacle for further structural and biochemical investigation. Based on the analysis of 43 eukaryotic membrane proteins, we present a cost-effective high-throughput approach for rapidly screening membrane proteins that can be overproduced to levels of >1 mg per liter in Saccharomyces cerevisiae. We find that 70% of the well expressed membrane proteins tested in this system are stable, targeted to the correct organelle, and monodisperse in either Fos-choline 12 (FC-12) or n-dodecyl-beta-D-maltoside. We illustrate the advantage of such an approach, with the purification of monodisperse human and yeast nucleotide-sugar transporters to unprecedented levels. We estimate that our approach should be able to provide milligram quantities for at least one-quarter of all membrane proteins from both yeast and higher eukaryotic organisms.

    PMID:
    17709746
    [PubMed - indexed for MEDLINE]
    PMCID: PMC1955786
    Free PMC Article

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