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Rapid Purification and Characterization of Mutant Origin Recognition Complexes in Saccharomyces cerevisiae.

Kawakami H, Ohashi E, Tsurimoto T, Katayama T - Front Microbiol (2016)

Bottom Line: All the six subunits of ORC are overexpressed at a considerable level and isolated as a functional heterohexameric complex.Furthermore, use of mammalian cells prevents contamination of wild-type ORC from yeast cells.The rapid acquisition of mutant ORCs using this system will boost systematic biochemical dissection of ORC and can be even applied to the purification of protein complexes other than ORC.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, Graduate School of Pharmaceutical Sciences, Kyushu University Fukuoka, Japan.

ABSTRACT
Purification of the origin recognition complex (ORC) from wild-type budding yeast cells more than two decades ago opened up doors to analyze the initiation of eukaryotic chromosomal DNA replication biochemically. Although revised methods to purify ORC from overproducing cells were reported later, purification of mutant proteins using these systems still depends on time-consuming processes including genetic manipulation to construct and amplify mutant baculoviruses or yeast strains as well as several canonical protein fractionations. Here, we present a streamlined method to construct mutant overproducers, followed by purification of mutant ORCs. Use of mammalian cells co-transfected with conveniently mutagenized plasmids bearing a His tag excludes many of the construction and fractionation steps. Transfection is highly efficient. All the six subunits of ORC are overexpressed at a considerable level and isolated as a functional heterohexameric complex. Furthermore, use of mammalian cells prevents contamination of wild-type ORC from yeast cells. The method is applicable to wild-type and at least three mutant ORCs, and the resultant purified complexes show expected biochemical activities. The rapid acquisition of mutant ORCs using this system will boost systematic biochemical dissection of ORC and can be even applied to the purification of protein complexes other than ORC.

No MeSH data available.


Related in: MedlinePlus

Workflow of overexpression and purification of mutant ORCs using a mammalian expression system. See text for details.
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Figure 4: Workflow of overexpression and purification of mutant ORCs using a mammalian expression system. See text for details.

Mentions: To minimize and simplify the column chromatography steps during purification, we first established a purification method of Orc1–5, which takes only 2 days (Figure 4). Orc1/2/3/4/5 form a stable heteropentamer (Lee and Bell, 1997; Chen et al., 2008). The method to purify Orc1–5 from cells using 40 15-cm plates is detailed below.


Rapid Purification and Characterization of Mutant Origin Recognition Complexes in Saccharomyces cerevisiae.

Kawakami H, Ohashi E, Tsurimoto T, Katayama T - Front Microbiol (2016)

Workflow of overexpression and purification of mutant ORCs using a mammalian expression system. See text for details.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4834435&req=5

Figure 4: Workflow of overexpression and purification of mutant ORCs using a mammalian expression system. See text for details.
Mentions: To minimize and simplify the column chromatography steps during purification, we first established a purification method of Orc1–5, which takes only 2 days (Figure 4). Orc1/2/3/4/5 form a stable heteropentamer (Lee and Bell, 1997; Chen et al., 2008). The method to purify Orc1–5 from cells using 40 15-cm plates is detailed below.

Bottom Line: All the six subunits of ORC are overexpressed at a considerable level and isolated as a functional heterohexameric complex.Furthermore, use of mammalian cells prevents contamination of wild-type ORC from yeast cells.The rapid acquisition of mutant ORCs using this system will boost systematic biochemical dissection of ORC and can be even applied to the purification of protein complexes other than ORC.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, Graduate School of Pharmaceutical Sciences, Kyushu University Fukuoka, Japan.

ABSTRACT
Purification of the origin recognition complex (ORC) from wild-type budding yeast cells more than two decades ago opened up doors to analyze the initiation of eukaryotic chromosomal DNA replication biochemically. Although revised methods to purify ORC from overproducing cells were reported later, purification of mutant proteins using these systems still depends on time-consuming processes including genetic manipulation to construct and amplify mutant baculoviruses or yeast strains as well as several canonical protein fractionations. Here, we present a streamlined method to construct mutant overproducers, followed by purification of mutant ORCs. Use of mammalian cells co-transfected with conveniently mutagenized plasmids bearing a His tag excludes many of the construction and fractionation steps. Transfection is highly efficient. All the six subunits of ORC are overexpressed at a considerable level and isolated as a functional heterohexameric complex. Furthermore, use of mammalian cells prevents contamination of wild-type ORC from yeast cells. The method is applicable to wild-type and at least three mutant ORCs, and the resultant purified complexes show expected biochemical activities. The rapid acquisition of mutant ORCs using this system will boost systematic biochemical dissection of ORC and can be even applied to the purification of protein complexes other than ORC.

No MeSH data available.


Related in: MedlinePlus