title: Leishmania tarentolae as a host for heterologous expression of functional human ABCB6 transporter.
abstract: The need for large amounts of reproducibly produced and isolated protein arises not only in structural studies, but even more so in biochemical ones, and with regard to ABC transporters it is especially pressing when faced with the prospect of enzymatic/transport activity studies, substrate screening etc. Thus, reliable heterologous expression systems/model organisms for large and complex proteins are at a premium. We have verified the applicability of the recently established novel eukaryotic expression system, using Leishmania tarentolae as a host, for human ABC protein overexpression. We succeeded in overexpressing human ABCB6, a protein with controversial subcellular localization and multiple proposed cellular functions. We were able to demonstrate its efficient expression in the expected subcellular locations as well as biochemical activity of the overexpressed protein (ATPase activity and porphyrin-like substrate transport). This activity was absent in cells overexpressing the catalytically inactive variant of ABCB6 (K629M). We demonstrate the possibility of applying a cost-effective expression system to study the activity of membrane transporters from the ABC superfamily.
authors.affiliation: Department of Molecular Biophysics, Faculty of Biology and Environmental Protection, University of Lodz, 12/16 Banacha St., 90-237 Lodz, Poland.
authors.forename: Jacek
authors.initials: J
authors.lastname: Grebowski
authors.affiliation: Department of Molecular Biophysics, Faculty of Biology and Environmental Protection, University of Lodz, 12/16 Banacha St., 90-237 Lodz, Poland.
authors.forename: Maciej
authors.initials: M
authors.lastname: Studzian
authors.affiliation: Department of Molecular Biophysics, Faculty of Biology and Environmental Protection, University of Lodz, 12/16 Banacha St., 90-237 Lodz, Poland.
authors.forename: Grzegorz
authors.initials: G
authors.lastname: Bartosz
authors.affiliation: Department of Molecular Biophysics, Faculty of Biology and Environmental Protection, University of Lodz, 12/16 Banacha St., 90-237 Lodz, Poland. Electronic address: lpulaski@uni.lodz.pl.
authors.forename: Lukasz
authors.initials: L
authors.lastname: Pulaski
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000096:biosynthesis / Q000235:genetics*; D001483:Base Sequence; D001692:Biological Transport; D017931:DNA Primers / Q000737:chemistry; D057075:Enzyme Assays; D015870:Gene Expression; D005822:Genetic Vectors / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D007425:Intracellular Membranes / Q000737:chemistry / Q000378:metabolism; D007891:Leishmania / Q000166:cytology / Q000235:genetics* / Q000378:metabolism; D008861:Microsomes / Q000737:chemistry / Q000378:metabolism; D009154:Mutation; D030781:Organisms, Genetically Modified; D010710:Phosphates / Q000737:chemistry* / Q000378:metabolism; D011166:Porphyrins / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D013379:Substrate Specificity
chemical_list: C107991:ABCB6 protein, human; D018528:ATP-Binding Cassette Transporters; D017931:DNA Primers; D010710:Phosphates; D011166:Porphyrins; D011994:Recombinant Proteins
keywords: ABC transporters; Leishmania; Membrane protein expression