Heterologous overexpression and mutagenesis of the human bile salt export pump (ABCB11) using DREAM (Directed REcombination-Assisted Mutagenesis).
basic_science · Level V
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- Record sourced from PubMed, PMID 21655228.
- Also identified by DOI 10.1371/journal.pone.0020562 and PMC identifier PMC2278221.
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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.