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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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Heterologous overexpression of BSEP in Saccharomyces cerevisiae and Pichia pastoris.A, Toxic or unstable expression plasmids can be constructed for any system in S. cerevisiae by adding the necessary sequence to the plasmid backbone. In order to clone BSEP into the Pichia pastoris expression cassette on pPIC3.5, the recombination vector was double-digested to allow the simultaneous insertion of both the unstable BSEP coding sequence and a PCR-generated fragment of the YEpHIS plasmid carrying the 2 micron origin (Ori) of replication and the leucine (LEU) prototrophy marker by homologous recombination (RS = NdeI). This plasmid was recovered from S. cerevisiae and obtained in preparative amounts from E. coli by strict cultivation at 30°C under suitable conditions. B, Expression of human BSEP in S. cerevisiae and P. pastoris. Equal amounts of whole yeast cell extracts were resolved on SDS-PAGE, electroblotted and probed with the polyclonal BSEP antiserum K168. Left panel, Homologous recombination was used to construct both BSEP expression vectors directly in S. cerevisiae. NHIS/CHIS, N- or C-terminal his tag position; empty Ctrl, strain transformed with corresponding empty YEpHIS expression plasmid. Right panel, pPIC3.5-CHISBSEP was constructed as described in A and used to transform P. pastoris strain GS-115 by electroporation. Empty Ctrl, P. pastoris GS-115 strain transformed with the empty pPIC3.5 integration vector.
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pone-0020562-g001: Heterologous overexpression of BSEP in Saccharomyces cerevisiae and Pichia pastoris.A, Toxic or unstable expression plasmids can be constructed for any system in S. cerevisiae by adding the necessary sequence to the plasmid backbone. In order to clone BSEP into the Pichia pastoris expression cassette on pPIC3.5, the recombination vector was double-digested to allow the simultaneous insertion of both the unstable BSEP coding sequence and a PCR-generated fragment of the YEpHIS plasmid carrying the 2 micron origin (Ori) of replication and the leucine (LEU) prototrophy marker by homologous recombination (RS = NdeI). This plasmid was recovered from S. cerevisiae and obtained in preparative amounts from E. coli by strict cultivation at 30°C under suitable conditions. B, Expression of human BSEP in S. cerevisiae and P. pastoris. Equal amounts of whole yeast cell extracts were resolved on SDS-PAGE, electroblotted and probed with the polyclonal BSEP antiserum K168. Left panel, Homologous recombination was used to construct both BSEP expression vectors directly in S. cerevisiae. NHIS/CHIS, N- or C-terminal his tag position; empty Ctrl, strain transformed with corresponding empty YEpHIS expression plasmid. Right panel, pPIC3.5-CHISBSEP was constructed as described in A and used to transform P. pastoris strain GS-115 by electroporation. Empty Ctrl, P. pastoris GS-115 strain transformed with the empty pPIC3.5 integration vector.

Mentions: The unicelluar eukaryote S. cerevisiae was initially chosen because of three advantages: (i) it can perform efficient homologous recombination [5], [7]; (ii) expression of other eukaryotic ABC transporters has been successfully reported [10]. For example, S. cerevisiae has been used to express the BSEP homologue MDR1 [11], [12]. (iii) Transformants resulting from in vivo homologous recombination can immediately be tested for target protein expression. We used these advantages for BSEP, but expression levels in S. cerevisiae were very low and not sufficient for subsequent purification or activity studies (Figure 1B, left panel).


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)

Heterologous overexpression of BSEP in Saccharomyces cerevisiae and Pichia pastoris.A, Toxic or unstable expression plasmids can be constructed for any system in S. cerevisiae by adding the necessary sequence to the plasmid backbone. In order to clone BSEP into the Pichia pastoris expression cassette on pPIC3.5, the recombination vector was double-digested to allow the simultaneous insertion of both the unstable BSEP coding sequence and a PCR-generated fragment of the YEpHIS plasmid carrying the 2 micron origin (Ori) of replication and the leucine (LEU) prototrophy marker by homologous recombination (RS = NdeI). This plasmid was recovered from S. cerevisiae and obtained in preparative amounts from E. coli by strict cultivation at 30°C under suitable conditions. B, Expression of human BSEP in S. cerevisiae and P. pastoris. Equal amounts of whole yeast cell extracts were resolved on SDS-PAGE, electroblotted and probed with the polyclonal BSEP antiserum K168. Left panel, Homologous recombination was used to construct both BSEP expression vectors directly in S. cerevisiae. NHIS/CHIS, N- or C-terminal his tag position; empty Ctrl, strain transformed with corresponding empty YEpHIS expression plasmid. Right panel, pPIC3.5-CHISBSEP was constructed as described in A and used to transform P. pastoris strain GS-115 by electroporation. Empty Ctrl, P. pastoris GS-115 strain transformed with the empty pPIC3.5 integration vector.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3105083&req=5

pone-0020562-g001: Heterologous overexpression of BSEP in Saccharomyces cerevisiae and Pichia pastoris.A, Toxic or unstable expression plasmids can be constructed for any system in S. cerevisiae by adding the necessary sequence to the plasmid backbone. In order to clone BSEP into the Pichia pastoris expression cassette on pPIC3.5, the recombination vector was double-digested to allow the simultaneous insertion of both the unstable BSEP coding sequence and a PCR-generated fragment of the YEpHIS plasmid carrying the 2 micron origin (Ori) of replication and the leucine (LEU) prototrophy marker by homologous recombination (RS = NdeI). This plasmid was recovered from S. cerevisiae and obtained in preparative amounts from E. coli by strict cultivation at 30°C under suitable conditions. B, Expression of human BSEP in S. cerevisiae and P. pastoris. Equal amounts of whole yeast cell extracts were resolved on SDS-PAGE, electroblotted and probed with the polyclonal BSEP antiserum K168. Left panel, Homologous recombination was used to construct both BSEP expression vectors directly in S. cerevisiae. NHIS/CHIS, N- or C-terminal his tag position; empty Ctrl, strain transformed with corresponding empty YEpHIS expression plasmid. Right panel, pPIC3.5-CHISBSEP was constructed as described in A and used to transform P. pastoris strain GS-115 by electroporation. Empty Ctrl, P. pastoris GS-115 strain transformed with the empty pPIC3.5 integration vector.
Mentions: The unicelluar eukaryote S. cerevisiae was initially chosen because of three advantages: (i) it can perform efficient homologous recombination [5], [7]; (ii) expression of other eukaryotic ABC transporters has been successfully reported [10]. For example, S. cerevisiae has been used to express the BSEP homologue MDR1 [11], [12]. (iii) Transformants resulting from in vivo homologous recombination can immediately be tested for target protein expression. We used these advantages for BSEP, but expression levels in S. cerevisiae were very low and not sufficient for subsequent purification or activity studies (Figure 1B, left panel).

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