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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 ORC containing Orc1 K362A. 293T cells co-overexpressing Orc1 K362A-His, Orc2, Orc3, Orc4, Orc5, and Orc6 were lyzed and fractionated using HisTrap (A) and SP Sepharose and Superdex 200 columns (B). The indicated volume was taken and analyzed using 9% SDS-PAGE, followed by Coomassie staining. The migration of each ORC subunit is indicated.
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Figure 6: Purification of ORC containing Orc1 K362A. 293T cells co-overexpressing Orc1 K362A-His, Orc2, Orc3, Orc4, Orc5, and Orc6 were lyzed and fractionated using HisTrap (A) and SP Sepharose and Superdex 200 columns (B). The indicated volume was taken and analyzed using 9% SDS-PAGE, followed by Coomassie staining. The migration of each ORC subunit is indicated.

Mentions: We next attempted to purify mutant ORC hexamers. This time, we purified ORC containing His-tagged Orc1 K362A from cells using 20 15-cm plates, half the number that was used for Orc1–5. This time we substituted HisTrap column chromatography for MagneHis (Figure 4) to perform a linear gradient elution. The revised method takes only 1 week, including the DNA work such as site-directed mutagenesis. Orc1 K362A-His and Orc1 R367A-His were overexpressed in 293T cells as soluble proteins, similar to wild-type Orc1 (Supplementary Figure 1). When HisTrap column chromatography was performed, Orc1 K362A-His was eluted relatively broadly, peaking at fraction numbers 19–21 (Figure 6A). Some major proteins corresponding to Orc2/3/4/5/6 co-migrated slightly slower, peaking at fraction numbers 23 and 24 (Figure 6A), suggesting that His-tagged Orc1 K362A and His-tagged Orc1 K362A containing Orc2/3/4/5/6 eluted at slightly different imidazole concentrations. Fractions containing all of the ORC subunits were pooled, concentrated, and further fractionated by gel filtration. As expected, His-tagged Orc1-K362A was separated into two fractions, the faster co-migrated with Orc2/3/4/5/6 and the slower eluted alone (Figure 6B). Each band was nearly stoichiometric, suggesting that His-tagged Orc1 K362A as well as Orc2/3/4/5/6 formed a stoichiometric hexamer. Similar results were obtained during preparation of wild-type ORC and ORC containing His-tagged Orc1 R367A; ~0.4–0.7 mg of purified ORC was yielded under these conditions, which is sufficient for typical biochemical assays (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 ORC containing Orc1 K362A. 293T cells co-overexpressing Orc1 K362A-His, Orc2, Orc3, Orc4, Orc5, and Orc6 were lyzed and fractionated using HisTrap (A) and SP Sepharose and Superdex 200 columns (B). The indicated volume was taken and analyzed using 9% SDS-PAGE, followed by Coomassie staining. The migration of each ORC subunit is indicated.
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Figure 6: Purification of ORC containing Orc1 K362A. 293T cells co-overexpressing Orc1 K362A-His, Orc2, Orc3, Orc4, Orc5, and Orc6 were lyzed and fractionated using HisTrap (A) and SP Sepharose and Superdex 200 columns (B). The indicated volume was taken and analyzed using 9% SDS-PAGE, followed by Coomassie staining. The migration of each ORC subunit is indicated.
Mentions: We next attempted to purify mutant ORC hexamers. This time, we purified ORC containing His-tagged Orc1 K362A from cells using 20 15-cm plates, half the number that was used for Orc1–5. This time we substituted HisTrap column chromatography for MagneHis (Figure 4) to perform a linear gradient elution. The revised method takes only 1 week, including the DNA work such as site-directed mutagenesis. Orc1 K362A-His and Orc1 R367A-His were overexpressed in 293T cells as soluble proteins, similar to wild-type Orc1 (Supplementary Figure 1). When HisTrap column chromatography was performed, Orc1 K362A-His was eluted relatively broadly, peaking at fraction numbers 19–21 (Figure 6A). Some major proteins corresponding to Orc2/3/4/5/6 co-migrated slightly slower, peaking at fraction numbers 23 and 24 (Figure 6A), suggesting that His-tagged Orc1 K362A and His-tagged Orc1 K362A containing Orc2/3/4/5/6 eluted at slightly different imidazole concentrations. Fractions containing all of the ORC subunits were pooled, concentrated, and further fractionated by gel filtration. As expected, His-tagged Orc1-K362A was separated into two fractions, the faster co-migrated with Orc2/3/4/5/6 and the slower eluted alone (Figure 6B). Each band was nearly stoichiometric, suggesting that His-tagged Orc1 K362A as well as Orc2/3/4/5/6 formed a stoichiometric hexamer. Similar results were obtained during preparation of wild-type ORC and ORC containing His-tagged Orc1 R367A; ~0.4–0.7 mg of purified ORC was yielded under these conditions, which is sufficient for typical biochemical assays (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