title: Functional Expression and Characterization of Plant ABC Transporters in Xenopus laevis Oocytes for Transport Engineering Purposes.
abstract: Transport engineering in bioengineering is aimed at efficient export of the final product to reduce toxicity and feedback inhibition and to increase yield. The ATP-binding cassette (ABC) transporters with their highly diverse substrate specificity and role in cellular efflux are potentially suitable in transport engineering approaches, although their size and high number of introns make them notoriously difficult to clone. Here, we report a novel in planta "exon engineering" strategy for cloning of full-length coding sequence of ABC transporters followed by methods for biochemical characterization of ABC exporters in Xenopus oocytes. Although the Xenopus oocyte expression system is particularly suitable for expression of membrane proteins and powerful in screening for novel transporter activity, only few examples of successful expression of ABC transporter has been reported. This raises the question whether the oocytes system is suitable to express and characterize ABC transporters. Thus we have selected AtABCG25, previously characterized in insect cells as the exporter of commercially valuable abscisic acid-as case study for optimizing of characterization in Xenopus oocytes. The tools provided will hopefully contribute to more successful transport engineering in synthetic biology.
authors.affiliation: DynaMo Center, Faculty of Science, University of Copenhagen, Frederiksberg C, Denmark.
authors.forename: D
authors.initials: D
authors.lastname: Xu
authors.affiliation: DynaMo Center, Faculty of Science, University of Copenhagen, Frederiksberg C, Denmark.
authors.forename: D
authors.initials: D
authors.lastname: Veres
authors.affiliation: DynaMo Center, Faculty of Science, University of Copenhagen, Frederiksberg C, Denmark.
authors.forename: Z M
authors.initials: ZM
authors.lastname: Belew
authors.affiliation: Faculty of Science, University of Copenhagen, Frederiksberg C, Denmark.
authors.forename: C E
authors.initials: CE
authors.lastname: Olsen
authors.affiliation: DynaMo Center, Faculty of Science, University of Copenhagen, Frederiksberg C, Denmark.
authors.forename: H H
authors.initials: HH
authors.lastname: Nour-Eldin
authors.affiliation: DynaMo Center, Faculty of Science, University of Copenhagen, Frederiksberg C, Denmark. Electronic address: bah@plen.ku.dk.
authors.forename: B A
authors.initials: BA
authors.lastname: Halkier
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D017360:Arabidopsis / Q000235:genetics* / Q000378:metabolism; D029681:Arabidopsis Proteins / Q000235:genetics* / Q000378:metabolism; D057005:Bioengineering / Q000379:methods; D001692:Biological Transport; D003001:Cloning, Molecular / Q000379:methods*; D005091:Exons; D009865:Oocytes / Q000378:metabolism*; D013379:Substrate Specificity; D014982:Xenopus laevis / Q000235:genetics* / Q000378:metabolism
chemical_list: D018528:ATP-Binding Cassette Transporters; D029681:Arabidopsis Proteins
keywords: ABC transporter; Abscisic acid; Cloning of coding sequence; Exon engineering; Export assay; Functional transporter expression; Import assay; Transport engineering; Xenopus oocytes