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Heterologous overexpression and mutagenesis of the human bile salt export pump (ABCB11) using DREAM (Directed REcombination-Assisted Mutagenesis).

Stindt J, Ellinger P, Stross C, Keitel V, Häussinger D, Smits SH, Kubitz R, Schmitt L - PLoS ONE (2011)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biochemistry, Heinrich-Heine-University, Düsseldorf, Germany.

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.

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The yeast-maintainable BSEP expression plasmid is functionally indistinguishable from the non-yeast parental plasmid in mammalian cell culture.A, HEK293 cells were transfected with pEYFP-N1-BSEP. B, HEK293 cells were transfected with pEYFP-N1-OriLeu-BSEP. Equimolar amounts of both constructs showed comparable transfection efficiencies and expression levels (established by FACS analysis, see Figure S3). C, HEK293 (upper panel), MDCK (middle), and HepG2 cells (lower panel) were transfected with the BstBI mutation in pEYFP-N1-OriLeu-BSEP that was introduced with the E. coli-free site-directed mutagenesis method. All cells were transfected with equimolar amounts of the respective constructs via Lipofectamine according to the manufacturer's guidelines. After fixation, nuclei were stained with Hoechst 34580 (blue), and the fluorescence (green) of the YFP tag in the BSEP fusion protein was observed.
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pone-0020562-g003: The yeast-maintainable BSEP expression plasmid is functionally indistinguishable from the non-yeast parental plasmid in mammalian cell culture.A, HEK293 cells were transfected with pEYFP-N1-BSEP. B, HEK293 cells were transfected with pEYFP-N1-OriLeu-BSEP. Equimolar amounts of both constructs showed comparable transfection efficiencies and expression levels (established by FACS analysis, see Figure S3). C, HEK293 (upper panel), MDCK (middle), and HepG2 cells (lower panel) were transfected with the BstBI mutation in pEYFP-N1-OriLeu-BSEP that was introduced with the E. coli-free site-directed mutagenesis method. All cells were transfected with equimolar amounts of the respective constructs via Lipofectamine according to the manufacturer's guidelines. After fixation, nuclei were stained with Hoechst 34580 (blue), and the fluorescence (green) of the YFP tag in the BSEP fusion protein was observed.

Mentions: Many eukaryotic membrane proteins are expressed and studied in mammalian cell lines [27], [28]. In order to extend the usage of the DREAM method to human cell culture, a yeast-compatible derivative of the mammalian BSEP expression vector pEYFP-N1-BSEP [29] (Figure S1) was created by introducing the necessary S. cerevisiae ORI and selection marker(in analogy to the P. pastoris vector modification shown in Figure 1A). After introduction and sequence verification, the obtained plasmid construct was successfully transfected into HEK293 cells as visualized by detection of the fluorescent YFP tag at the C-terminus of the BSEP fusion protein (Figure 3). Comparison with the parental non-yeast expression vector showed that in both cases BSEP is localized at the plasma membrane (Figure 3A and B). Additionally, flow cytometric analyses of HEK293 cells transfected with equimolar amounts of BSEP in pEYFP-N1 and in pEYFP-N1-OriLeu, respectively, were performed to determine transfection and expression rates of both plasmids (Figure S3). Here, transfection rates and BSEP expression from the larger yeast shuttle vector and the smaller, parental vector were found to be comparable (21% and 32%, respectively). These experiments, show that the presence of the additional ORI and selection marker does not influence the expression of BSEP in mammalian cell cultures.


Heterologous overexpression and mutagenesis of the human bile salt export pump (ABCB11) using DREAM (Directed REcombination-Assisted Mutagenesis).

Stindt J, Ellinger P, Stross C, Keitel V, Häussinger D, Smits SH, Kubitz R, Schmitt L - PLoS ONE (2011)

The yeast-maintainable BSEP expression plasmid is functionally indistinguishable from the non-yeast parental plasmid in mammalian cell culture.A, HEK293 cells were transfected with pEYFP-N1-BSEP. B, HEK293 cells were transfected with pEYFP-N1-OriLeu-BSEP. Equimolar amounts of both constructs showed comparable transfection efficiencies and expression levels (established by FACS analysis, see Figure S3). C, HEK293 (upper panel), MDCK (middle), and HepG2 cells (lower panel) were transfected with the BstBI mutation in pEYFP-N1-OriLeu-BSEP that was introduced with the E. coli-free site-directed mutagenesis method. All cells were transfected with equimolar amounts of the respective constructs via Lipofectamine according to the manufacturer's guidelines. After fixation, nuclei were stained with Hoechst 34580 (blue), and the fluorescence (green) of the YFP tag in the BSEP fusion protein was observed.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3105083&req=5

pone-0020562-g003: The yeast-maintainable BSEP expression plasmid is functionally indistinguishable from the non-yeast parental plasmid in mammalian cell culture.A, HEK293 cells were transfected with pEYFP-N1-BSEP. B, HEK293 cells were transfected with pEYFP-N1-OriLeu-BSEP. Equimolar amounts of both constructs showed comparable transfection efficiencies and expression levels (established by FACS analysis, see Figure S3). C, HEK293 (upper panel), MDCK (middle), and HepG2 cells (lower panel) were transfected with the BstBI mutation in pEYFP-N1-OriLeu-BSEP that was introduced with the E. coli-free site-directed mutagenesis method. All cells were transfected with equimolar amounts of the respective constructs via Lipofectamine according to the manufacturer's guidelines. After fixation, nuclei were stained with Hoechst 34580 (blue), and the fluorescence (green) of the YFP tag in the BSEP fusion protein was observed.
Mentions: Many eukaryotic membrane proteins are expressed and studied in mammalian cell lines [27], [28]. In order to extend the usage of the DREAM method to human cell culture, a yeast-compatible derivative of the mammalian BSEP expression vector pEYFP-N1-BSEP [29] (Figure S1) was created by introducing the necessary S. cerevisiae ORI and selection marker(in analogy to the P. pastoris vector modification shown in Figure 1A). After introduction and sequence verification, the obtained plasmid construct was successfully transfected into HEK293 cells as visualized by detection of the fluorescent YFP tag at the C-terminus of the BSEP fusion protein (Figure 3). Comparison with the parental non-yeast expression vector showed that in both cases BSEP is localized at the plasma membrane (Figure 3A and B). Additionally, flow cytometric analyses of HEK293 cells transfected with equimolar amounts of BSEP in pEYFP-N1 and in pEYFP-N1-OriLeu, respectively, were performed to determine transfection and expression rates of both plasmids (Figure S3). Here, transfection rates and BSEP expression from the larger yeast shuttle vector and the smaller, parental vector were found to be comparable (21% and 32%, respectively). These experiments, show that the presence of the additional ORI and selection marker does not influence the expression of BSEP in mammalian cell cultures.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biochemistry, Heinrich-Heine-University, Düsseldorf, Germany.

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.

Show MeSH
Related in: MedlinePlus