title: Expression and purification of homogenous proteins in Saccharomyces cerevisiae based on ubiquitin-FLAG fusion.
abstract: The construction of an expression vector for increased expression of cytoplasmic proteins in Saccharomyces cerevisiae is described. To enhance the yield of expressed proteins, fusion of ubiquitin to an octapeptide (a FLAG tag) upstream of the respective model genes was applied. During protein maturation ubiquitin is efficiently removed by yeast autologous hydrolases, generating the FLAG octapeptide at the N-terminus. Fusion proteins were recognized by the specific monoclonal antibody M1 directed against the FLAG tag. The FLAG-tagged proteins were purified to homogeneity by immunoaffinity chromatography using an anti-FLAG M1 agarose. Different model proteins, green fluorescent protein, green fluorescent protein-human lysozyme, green fluorescent protein elongation-initiahon factor 5a, green fluorescent protein-rapamycin-selective 25-kDa immunophilin, and green fluorescent protein-heat shock protein 90 beta have been selected to demonstrate the efficiency of the new vector construct.
authors.affiliation: Institute for Applied Microbiology, University of Agricultural Sciences, Muthgasse 18, 1190 Vienna, Austria.
authors.forename: A
authors.initials: A
authors.lastname: Einhauer
authors.forename: M
authors.initials: M
authors.lastname: Schuster
authors.forename: E
authors.initials: E
authors.lastname: Wasserbauer
authors.forename: A
authors.initials: A
authors.lastname: Jungbauer
mesh_terms: D000911:Antibodies, Monoclonal; D015153:Blotting, Western; D002846:Chromatography, Affinity; D009842:Oligopeptides; D010455:Peptides / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae; D025801:Ubiquitin / Q000235:genetics* / Q000302:isolation & purification
chemical_list: D000911:Antibodies, Monoclonal; D009842:Oligopeptides; D010455:Peptides; D011993:Recombinant Fusion Proteins; D025801:Ubiquitin; C085706:FLAG peptide