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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 DREAM method allows site-directed mutagenesis of an unstable BSEP-carrying plasmid without the need for E. coli.A, The product yield can be increased significantly by shifting the primer binding sites from a complete to a partial overlap that allows binding of the primers to product in subsequent cycles. The minute amount (10 ng) of non-mutated template used in the reaction is easily outnumbered by the yield of mutagenesis product, which is double-stranded (ds) due to the modified primer design (see also Figure S2). This allows for the direct transformation of S. cerevisiae without the need of prior template removal. The mutagenesis product is then recircularized by homologous recombination of the double-stranded ends. B, Analysis of S. cerevisisae transformants obtained from the modified SDM protocol shows a high mutagenesis efficiency. Successful mutagenesis in this case results in the addition of an BstBI restriction site into the BSEP coding sequence. Colony PCR of the resulting transformants was performed with primers surrounding the mutagenesis site, and the resulting product was digested with BstBI. 19 of the 25 tested clones carried the additional restriction site (+), corresponding to a mutagenesis efficiency of 76%.
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pone-0020562-g002: The DREAM method allows site-directed mutagenesis of an unstable BSEP-carrying plasmid without the need for E. coli.A, The product yield can be increased significantly by shifting the primer binding sites from a complete to a partial overlap that allows binding of the primers to product in subsequent cycles. The minute amount (10 ng) of non-mutated template used in the reaction is easily outnumbered by the yield of mutagenesis product, which is double-stranded (ds) due to the modified primer design (see also Figure S2). This allows for the direct transformation of S. cerevisiae without the need of prior template removal. The mutagenesis product is then recircularized by homologous recombination of the double-stranded ends. B, Analysis of S. cerevisisae transformants obtained from the modified SDM protocol shows a high mutagenesis efficiency. Successful mutagenesis in this case results in the addition of an BstBI restriction site into the BSEP coding sequence. Colony PCR of the resulting transformants was performed with primers surrounding the mutagenesis site, and the resulting product was digested with BstBI. 19 of the 25 tested clones carried the additional restriction site (+), corresponding to a mutagenesis efficiency of 76%.

Mentions: Classic SDM relies on the removal of non-mutated template plasmid achieved by DpnI digestion, which recognizes and cleaves only methylated DNA template. Thereby only the mutated plasmid can give colonies. Plasmids prepared directly from S. cerevisiae, however are, unmethylated and the template plasmid cannot be removed by DpnI digestion [19], [20], [21]. Thereby, the efficiency of mutagenesis is too low. To obtain positive clones, several hundred nanograms of mutated plasmid are needed and a substantial amount of time is required to pick and analyze several clones to find the correctly mutated plasmid [22], [23], [24]. By changing the mutagenesis primer design from a complete to a partial, 5′-overlap of the primer pair (Figure 2A, step 1), the linear in vitro mutagenesis step is turned into an exponential polymerase chain reaction: due to this primer shift a product is generated which carries priming sites that serve as a template in the subsequent reaction cycles (step 2). The usefulness of such a primer shift was previously reported, although in a different context [25]. The reaction product is therefore endowed with homologous double-stranded ends that allow the precise recirculation by homologous recombination into an intact plasmid (step 3; for a detailed comparison of both, classic and DREAM mutagenesis, see Figure S2). A change of primers to mutated plasmid DNA used for transformation thereby increases the probability of picking positive mutated clones.


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 DREAM method allows site-directed mutagenesis of an unstable BSEP-carrying plasmid without the need for E. coli.A, The product yield can be increased significantly by shifting the primer binding sites from a complete to a partial overlap that allows binding of the primers to product in subsequent cycles. The minute amount (10 ng) of non-mutated template used in the reaction is easily outnumbered by the yield of mutagenesis product, which is double-stranded (ds) due to the modified primer design (see also Figure S2). This allows for the direct transformation of S. cerevisiae without the need of prior template removal. The mutagenesis product is then recircularized by homologous recombination of the double-stranded ends. B, Analysis of S. cerevisisae transformants obtained from the modified SDM protocol shows a high mutagenesis efficiency. Successful mutagenesis in this case results in the addition of an BstBI restriction site into the BSEP coding sequence. Colony PCR of the resulting transformants was performed with primers surrounding the mutagenesis site, and the resulting product was digested with BstBI. 19 of the 25 tested clones carried the additional restriction site (+), corresponding to a mutagenesis efficiency of 76%.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3105083&req=5

pone-0020562-g002: The DREAM method allows site-directed mutagenesis of an unstable BSEP-carrying plasmid without the need for E. coli.A, The product yield can be increased significantly by shifting the primer binding sites from a complete to a partial overlap that allows binding of the primers to product in subsequent cycles. The minute amount (10 ng) of non-mutated template used in the reaction is easily outnumbered by the yield of mutagenesis product, which is double-stranded (ds) due to the modified primer design (see also Figure S2). This allows for the direct transformation of S. cerevisiae without the need of prior template removal. The mutagenesis product is then recircularized by homologous recombination of the double-stranded ends. B, Analysis of S. cerevisisae transformants obtained from the modified SDM protocol shows a high mutagenesis efficiency. Successful mutagenesis in this case results in the addition of an BstBI restriction site into the BSEP coding sequence. Colony PCR of the resulting transformants was performed with primers surrounding the mutagenesis site, and the resulting product was digested with BstBI. 19 of the 25 tested clones carried the additional restriction site (+), corresponding to a mutagenesis efficiency of 76%.
Mentions: Classic SDM relies on the removal of non-mutated template plasmid achieved by DpnI digestion, which recognizes and cleaves only methylated DNA template. Thereby only the mutated plasmid can give colonies. Plasmids prepared directly from S. cerevisiae, however are, unmethylated and the template plasmid cannot be removed by DpnI digestion [19], [20], [21]. Thereby, the efficiency of mutagenesis is too low. To obtain positive clones, several hundred nanograms of mutated plasmid are needed and a substantial amount of time is required to pick and analyze several clones to find the correctly mutated plasmid [22], [23], [24]. By changing the mutagenesis primer design from a complete to a partial, 5′-overlap of the primer pair (Figure 2A, step 1), the linear in vitro mutagenesis step is turned into an exponential polymerase chain reaction: due to this primer shift a product is generated which carries priming sites that serve as a template in the subsequent reaction cycles (step 2). The usefulness of such a primer shift was previously reported, although in a different context [25]. The reaction product is therefore endowed with homologous double-stranded ends that allow the precise recirculation by homologous recombination into an intact plasmid (step 3; for a detailed comparison of both, classic and DREAM mutagenesis, see Figure S2). A change of primers to mutated plasmid DNA used for transformation thereby increases the probability of picking positive mutated clones.

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