title: Heterologous overexpression and mutagenesis of the human bile salt export pump (ABCB11) using DREAM (Directed REcombination-Assisted Mutagenesis).
abstract: Homologous recombination in Saccharomyces cerevisiae is a well-studied process. Here, we describe a yeast-recombination-based approach to construct and mutate plasmids containing the cDNA of the human bile salt export pump (BSEP) that has been shown to be unstable in E. coli. Using this approach, we constructed the necessary plasmids for a heterologous overexpression of BSEP in the yeast Pichia pastoris. We then applied a new site-directed mutagenesis method, DREAM (Directed REcombination-Assisted Mutagenesis) that completely bypasses E. coli by using S. cerevisiae as the plasmid host with high mutagenesis efficiency. Finally, we show how to apply this strategy to unstable non-yeast plasmids by rapidly turning an existing mammalian BSEP expression construct into a S. cerevisiae-compatible plasmid and analyzing the impact of a BSEP mutation in several mammalian cell lines.
authors.affiliation: Institute of Biochemistry, Heinrich-Heine-University, Düsseldorf, Germany.
authors.forename: Jan
authors.initials: J
authors.lastname: Stindt
authors.forename: Philipp
authors.initials: P
authors.lastname: Ellinger
authors.forename: Claudia
authors.initials: C
authors.lastname: Stross
authors.forename: Verena
authors.initials: V
authors.lastname: Keitel
authors.forename: Dieter
authors.initials: D
authors.lastname: Häussinger
authors.forename: Sander H J
authors.initials: SH
authors.lastname: Smits
authors.forename: Ralf
authors.initials: R
authors.lastname: Kubitz
authors.forename: Lutz
authors.initials: L
authors.lastname: Schmitt
mesh_terms: D000074020:ATP Binding Cassette Transporter, Subfamily B, Member 11; D018528:ATP-Binding Cassette Transporters / Q000235:genetics / Q000378:metabolism*; D005434:Flow Cytometry; D057809:HEK293 Cells; D006801:Humans; D016297:Mutagenesis, Site-Directed; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism
chemical_list: C093491:ABCB11 protein, human; D000074020:ATP Binding Cassette Transporter, Subfamily B, Member 11; D018528:ATP-Binding Cassette Transporters