title: New examples of membrane protein expression and purification using the yeast based Pdr1-3 expression strategy.
abstract: Overexpression and purification of membrane proteins has been a bottleneck for their functional and structural study for a long time. Both homologous and heterologous expression of membrane proteins with suitable tags for purification presents unique challenges for cloning and expression. Saccharomyces cerevisiae is a potential host system with significant closeness to higher eukaryotes and provides opportunity for attempts to express membrane proteins. In the past, bakers yeast containing mutations within the transcriptional regulator Pdr1 has been used to overexpress various membrane proteins including for example the ABC transporters Pdr5 and Yor1, respectively. In this study we exploited this system and tried to express and purify 3 membrane proteins in yeast along with Pdr5 and Yor1 viz. Rsb1, Mdl1 and Drs2 by virtue of an N-terminal 14-histidine affinity tag. Out of these five, we could express all membrane proteins although at different levels. Satisfactory yields were obtained for three examples i.e. Pdr5, Yor1 and Drs2. Rsb1 expression was comparatively low and Mdl1 was rather unsatisfactory. Thus, we demonstrate here the application of this yeast based expression system that is suitable for cloning, expression and purification of a wide variety of membrane proteins.
authors.affiliation: Institute of Biochemistry, Heinrich Heine University Düsseldorf, Universitätsstr. 1, 40225 Düsseldorf, Germany.
authors.forename: Rakeshkumar P
authors.initials: RP
authors.lastname: Gupta
authors.affiliation: Institute of Biochemistry, Heinrich Heine University Düsseldorf, Universitätsstr. 1, 40225 Düsseldorf, Germany.
authors.forename: Petra
authors.initials: P
authors.lastname: Kueppers
authors.affiliation: Institute of Biochemistry, Heinrich Heine University Düsseldorf, Universitätsstr. 1, 40225 Düsseldorf, Germany. Electronic address: lutz.schmitt@hhu.de.
authors.forename: Lutz
authors.initials: L
authors.lastname: Schmitt
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D000252:Calcium-Transporting ATPases / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000096:biosynthesis* / Q000235:genetics; D015966:Gene Expression Regulation, Fungal; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D009154:Mutation; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D014157:Transcription Factors / Q000096:biosynthesis* / Q000235:genetics
chemical_list: D018528:ATP-Binding Cassette Transporters; D004268:DNA-Binding Proteins; C104014:DRS2 protein, S cerevisiae; C490069:MDL1 protein, S cerevisiae; D008565:Membrane Proteins; C085480:PDR1 protein, S cerevisiae; C092438:PDR5 protein, S cerevisiae; C465340:RSB1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; C097077:YOR1 protein, S cerevisiae; D000252:Calcium-Transporting ATPases
keywords: ATP binding cassette (ABC) transporters; Floppases; Membrane protein overexpression; Multidrug resistance; P-type ATPases