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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

Purification of Orc1–5. 293T cells co-overexpressing Orc1-His, Orc2, Orc3, Orc4, and Orc5 were lyzed and fractionated. The indicated volume was taken and analyzed using 9% SDS-PAGE, followed by Coomassie staining. The migration of each ORC subunit is indicated. W, whole cells; INP, input; FT, flow-through; W5, the fifth wash fraction; E, eluate; and 14–21, fraction numbers.
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Figure 5: Purification of Orc1–5. 293T cells co-overexpressing Orc1-His, Orc2, Orc3, Orc4, and Orc5 were lyzed and fractionated. The indicated volume was taken and analyzed using 9% SDS-PAGE, followed by Coomassie staining. The migration of each ORC subunit is indicated. W, whole cells; INP, input; FT, flow-through; W5, the fifth wash fraction; E, eluate; and 14–21, fraction numbers.

Mentions: Proteins at each step were monitored by SDS-PAGE (Figure 5). Most cellular proteins were soluble (lanes 1 and 2). Enrichment of a band at ~110 kDa was observed after MagneHis pulldown, corresponding to Orc1-His (106 kDa; lane 5). Some proteins including Orc2/3/4/5 were also enriched (lane 5). Orc1-His and Orc2/3/4/5 were concentrated by SP Sepharose (lane 7 and Table 1A). These proteins co-migrated during gel filtration (lanes 9–14), suggesting that they form a complex. The resultant ORC complex was purified to almost homogeneity. Concentration of the peak fractions yielded ~0.7 mg of protein, which is sufficient for most biochemical applications (Table 1).


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

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

Purification of Orc1–5. 293T cells co-overexpressing Orc1-His, Orc2, Orc3, Orc4, and Orc5 were lyzed and fractionated. The indicated volume was taken and analyzed using 9% SDS-PAGE, followed by Coomassie staining. The migration of each ORC subunit is indicated. W, whole cells; INP, input; FT, flow-through; W5, the fifth wash fraction; E, eluate; and 14–21, fraction numbers.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 5: Purification of Orc1–5. 293T cells co-overexpressing Orc1-His, Orc2, Orc3, Orc4, and Orc5 were lyzed and fractionated. The indicated volume was taken and analyzed using 9% SDS-PAGE, followed by Coomassie staining. The migration of each ORC subunit is indicated. W, whole cells; INP, input; FT, flow-through; W5, the fifth wash fraction; E, eluate; and 14–21, fraction numbers.
Mentions: Proteins at each step were monitored by SDS-PAGE (Figure 5). Most cellular proteins were soluble (lanes 1 and 2). Enrichment of a band at ~110 kDa was observed after MagneHis pulldown, corresponding to Orc1-His (106 kDa; lane 5). Some proteins including Orc2/3/4/5 were also enriched (lane 5). Orc1-His and Orc2/3/4/5 were concentrated by SP Sepharose (lane 7 and Table 1A). These proteins co-migrated during gel filtration (lanes 9–14), suggesting that they form a complex. The resultant ORC complex was purified to almost homogeneity. Concentration of the peak fractions yielded ~0.7 mg of protein, which is sufficient for most biochemical applications (Table 1).

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