title: Expression, purification and structural properties of ABC transporter ABCA4 and its individual domains.
abstract: ABCA4 is a member of the A subfamily of ATP-binding cassette transporters that consists of large integral membrane proteins implicated in inherited human diseases. ABCA4 assists in the clearance of N-retinylidene-phosphatidylethanolamine, a potentially toxic by-product of the visual cycle formed in photoreceptor cells during light perception. Structural and functional studies of this protein have been hindered by its large size, membrane association, and domain complexity. Although mammalian, insect and bacterial systems have been used for expression of ABCA4 and its individual domains, the structural relevance of resulting proteins to the native transporter has yet to be established. We produced soluble domains of ABCA4 in Escherichia coli and Saccharomyces cerevisiae and the full-length transporter in HEK293 cells. Electron microscopy and size exclusion chromatography were used to assess the conformational homogeneity and structure of these proteins. We found that isolated ABCA4 domains formed large, heterogeneous oligomers cross-linked with non-specific disulphide bonds. Incomplete folding of cytoplasmic domain 2 was proposed based on fluorescence spectroscopy results. In contrast, full-length human ABCA4 produced in mammalian cells was found structurally equivalent to the native protein obtained from bovine photoreceptors. These findings offer recombinantly expressed full-length ABCA4 as an appropriate object for future detailed structural and functional characterization.
authors.affiliation: Department of Pharmacology, School of Medicine, Case Western Reserve University, 10900 Euclid Ave, Cleveland, OH 44106, USA.
authors.forename: Yaroslav
authors.initials: Y
authors.lastname: Tsybovsky
authors.affiliation: Department of Pharmacology, School of Medicine, Case Western Reserve University, 10900 Euclid Ave, Cleveland, OH 44106, USA. Electronic address: kxp65@case.edu.
authors.forename: Krzysztof
authors.initials: K
authors.lastname: Palczewski
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification / Q000648:ultrastructure; D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D003001:Cloning, Molecular; D003545:Cysteine / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics; D057809:HEK293 Cells; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D058849:Protein Refolding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000648:ultrastructure; D012441:Saccharomyces cerevisiae / Q000235:genetics
chemical_list: C056475:ABCA4 protein, human; D018528:ATP-Binding Cassette Transporters; D004220:Disulfides; D011994:Recombinant Proteins; D003545:Cysteine
keywords: ABC transporter; ABCA subfamily; ABCA4; Electron microscopy; Heterologous expression; Monodispersity