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

Proc Natl Acad Sci U S A. 2007 Aug 28;104(35):13936-41. doi: 10.1073/pnas.0704546104. Epub 2007 Aug 20.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Gene Expression Regulation, Fungal
  • Genes, Reporter
  • Membrane Proteins / genetics
  • Membrane Proteins / isolation & purification
  • Membrane Proteins / metabolism*
  • Microscopy, Confocal
  • Plasmids
  • Recombinant Fusion Proteins / metabolism
  • Recombinant Proteins / genetics
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / metabolism
  • Saccharomyces cerevisiae / metabolism*
  • Saccharomyces cerevisiae Proteins / genetics
  • Saccharomyces cerevisiae Proteins / isolation & purification
  • Saccharomyces cerevisiae Proteins / metabolism

Substances

  • Membrane Proteins
  • Recombinant Fusion Proteins
  • Recombinant Proteins
  • Saccharomyces cerevisiae Proteins