GFP-based optimization scheme for the overexpression and purification of eukaryotic membrane proteins in Saccharomyces cerevisiae.
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.
Drew
David
D
Membrane Protein Crystallography Group, Division of Molecular Biosciences, Department of Life Sciences, Imperial College of London, London SW7 2AZ, UK.
Newstead
Simon
S
Sonoda
Yo
Y
Kim
Hyun
H
von Heijne
Gunnar
G
Iwata
So
S
D001709:Biotechnology / Q000379:methods*; D002850:Chromatography, Gel; D005822:Genetic Vectors / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000378:metabolism*; D008565:Membrane Proteins / Q000302:isolation & purification* / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000378:metabolism*
D008565:Membrane Proteins; D049452:Green Fluorescent Proteins