High-throughput fluorescent-based optimization of eukaryotic membrane protein overexpression and purification in <i>Saccharomyces cerevisiae</i>

DOI Web Site 被引用文献22件
  • Simon Newstead
    *Division of Molecular Biosciences, Membrane Protein Crystallography Group, Imperial College, London SW7 2AZ, United Kingdom;
  • Hyun Kim
    Center for Biomembrane Research, Department of Biochemistry and Biophysics, Stockholm University, SE-10691 Stockholm, Sweden;
  • Gunnar von Heijne
    Center for Biomembrane Research, Department of Biochemistry and Biophysics, Stockholm University, SE-10691 Stockholm, Sweden;
  • So Iwata
    *Division of Molecular Biosciences, Membrane Protein Crystallography Group, Imperial College, London SW7 2AZ, United Kingdom;
  • David Drew
    *Division of Molecular Biosciences, Membrane Protein Crystallography Group, Imperial College, London SW7 2AZ, United Kingdom;

説明

<jats:p> 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 <jats:italic>Saccharomyces cerevisiae</jats:italic> . 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 <jats:italic>n</jats:italic> -dodecyl-β- <jats:sc>d</jats:sc> -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. </jats:p>

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