title: Recombinant synthesis of human ABCG2 expressed in the yeast Saccharomyces cerevisiae: an experimental methodological study.
abstract: Human ABCG2 is an efflux protein belonging to the ATP-binding cassette transporter superfamily. It is expressed in the plasma membrane of different cell types performing various physiological functions. It is the most recently discovered MDR transporter and its structure and function are still not well understood. Thus, expression and functional reconstitution of the protein in different variants and from different sources are important steps for its further investigation. In this work we describe a recombinant synthesis of human ABCG2 R482G from S. cerevisiae. We expressed the human ABCG2 R482G variant in S. cerevisiae and purified the protein from total yeast membranes. Using a panel of sixteen detergents, we analyzed the efficiency of extraction of ABCG2 from membranes by SDS-PAGE and immunoblot analysis. Based on these results, three detergents were selected for further purification studies and two of them, n-octyl-β-D-glucopyranoside and n-dodecyl-β-D-maltopyranoside, yielded functional protein after reconstitution into liposomes. We show here the first example of purified and reconstituted ABCG2 expressed in S. cerevisiae retaining drug-stimulated ATPase activity.
authors.affiliation: Department of Pharmaceutical Chemistry, Institute of Pharmacy, University of Bonn, An der Immenburg 4, 53121 Bonn, Germany.
authors.forename: Anna
authors.initials: A
authors.lastname: Jacobs
authors.forename: Dana
authors.initials: D
authors.lastname: Emmert
authors.forename: Svenja
authors.initials: S
authors.lastname: Wieschrath
authors.forename: Christine A
authors.initials: CA
authors.lastname: Hrycyna
authors.forename: Michael
authors.initials: M
authors.lastname: Wiese
mesh_terms: D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D000595:Amino Acid Sequence; D005591:Chemical Fractionation / Q000379:methods*; D002845:Chromatography / Q000379:methods*; D015870:Gene Expression; D006801:Humans; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism
chemical_list: C116614:ABCG2 protein, human; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters; D009363:Neoplasm Proteins; D011994:Recombinant Proteins