Advanced method for high-throughput expression of mutated eukaryotic membrane proteins in Saccharomyces cerevisiae

Biochem Biophys Res Commun. 2008 Jul 11;371(4):841-5. doi: 10.1016/j.bbrc.2008.04.182. Epub 2008 May 12.


Crystallization of eukaryotic membrane proteins is a challenging, iterative process. The protein of interest is often modified in an attempt to improve crystallization and diffraction results. To accelerate this process, we took advantage of a GFP-fusion yeast expression system that uses PCR to direct homologous recombination and gene cloning. We explored the possibility of employing more than one PCR fragment to introduce various mutations in a single step, and found that when up to five PCR fragments were co-transformed into yeast, the recombination frequency was maintained as the number of fragments was increased. All transformants expressed the model membrane protein, while the resulting plasmid from each clone contained the designed mutations only. Thus, we have demonstrated a technique allowing the expression of mutant membrane proteins within 5 days, combining a GFP-fusion expression system and yeast homologous recombination.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cloning, Molecular / methods*
  • Green Fluorescent Proteins / genetics
  • Membrane Proteins / biosynthesis*
  • Membrane Proteins / genetics
  • Mice
  • Mutation
  • Polymerase Chain Reaction
  • Protein Biosynthesis
  • Recombinant Proteins / biosynthesis*
  • Recombinant Proteins / genetics
  • Recombination, Genetic
  • Saccharomyces cerevisiae / genetics*
  • TRPM Cation Channels / biosynthesis
  • TRPM Cation Channels / genetics
  • Transformation, Genetic


  • Membrane Proteins
  • Recombinant Proteins
  • TRPM Cation Channels
  • Trpm5 protein, mouse
  • Green Fluorescent Proteins