GFP-based optimization scheme for the overexpression and purification of eukaryotic membrane proteins in Saccharomyces cerevisiae.
2008 (English)In: Nat Protoc, ISSN 1750-2799, Vol. 3, no 5, 784-98 p.Article in journal (Refereed) Published
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 micro 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.
Place, publisher, year, edition, pages
2008. Vol. 3, no 5, 784-98 p.
Biotechnology/*methods, Chromatography; Gel, Genetic Vectors/genetics, Green Fluorescent Proteins/*metabolism, Membrane Proteins/*isolation & purification/*metabolism, Saccharomyces cerevisiae/*metabolism
IdentifiersURN: urn:nbn:se:su:diva-14921ISI: 000258423600004PubMedID: 18451787OAI: oai:DiVA.org:su-14921DiVA: diva2:181441