Functional Expression and Characterization of Plant ABC Transporters in Xenopus laevis Oocytes for Transport Engineering Purposes

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...

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Bibliographic Details
Published in:	Methods in enzymology Vol. 576; p. 207
Main Authors:	Xu, D, Veres, D, Belew, Z M, Olsen, C E, Nour-Eldin, H H, Halkier, B A
Format:	Journal Article
Language:	English
Published:	United States 2016
Subjects:	Animals Arabidopsis - genetics Arabidopsis - metabolism Arabidopsis Proteins - genetics Arabidopsis Proteins - metabolism ATP-Binding Cassette Transporters - genetics ATP-Binding Cassette Transporters - metabolism Bioengineering - methods Biological Transport Cloning, Molecular - methods Exons Oocytes - metabolism Substrate Specificity Xenopus laevis - genetics Xenopus laevis - metabolism Xenopus oocytes Cloning of coding sequence Abscisic acid Import assay Exon engineering ABC transporter Export assay Functional transporter expression Transport engineering
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Summary:	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.
ISSN:	1557-7988
DOI:	10.1016/bs.mie.2016.04.001