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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

Construction of ORC-overproducing plasmids. All the pHK plasmids used in this study are derivatives of ver. 3–5. See text for details.
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Figure 2: Construction of ORC-overproducing plasmids. All the pHK plasmids used in this study are derivatives of ver. 3–5. See text for details.

Mentions: To overproduce S. cerevisiae ORC in mammalian cells, we modified the ver. 3–5 vector for transfection, which was originally developed to overexpress proteins that are not overexpressed well in bacterial or insect cells (Figure 2; Uno and Masai, 2011; Uno et al., 2012). Ver. 3–5 is a shuttle vector carrying PEF−1α, a Kozak sequence, a His tag, a multiple cloning site, and an HA tag. Ver. 3–5 also bears an SV40 origin, which could help to maintain the vector episomally in mammalian cells expressing the large T antigen, such as 293T cells. To maintain the plasmid in Escherichia coli, ver. 3–5 also bears bla and ColE1 ori. We first cloned one of the ORC1/2/3/4/5/6 genes into ver. 3–5 so that the N-terminal His tag and C-terminal HA tag of the vector were eliminated, yielding pHK106 through to pHK111. By site-directed mutagenesis, a hexahistidine sequence with a linker was appended just before the stop codon of ORC1, yielding pHK118. This tag, consisting of 13 amino acids, is shorter than a CBP-TEV tag previously used for Orc1 tagging (Frigola et al., 2013). Addition of a short tag, such as His12 or His-Strep II, to the C-terminus of Orc1 does not affect Orc1 function in vivo (Kawakami et al., 2015). We noticed that introduction of certain orc1 mutations into pHK118 by site-directed mutagenesis was unsuccessful. Because the same mutation could be introduced into another ORC1 plasmid under the control of the native ORC1 promoter using the same mutagenic primers (Kawakami et al., 2015), one plausible idea is that leaky expression of Orc1 from pHK118 may be extremely toxic in E. coli cells only when a certain orc1 mutation is introduced. Because Orc1 solely binds to the ARS sequence via a domain termed EOS (Kawakami et al., 2015), similar binding to a similar sequence in the E. coli genome may be affected by the mutation and interfere with a certain cellular process in vivo. Alternatively, adverse interactions of the Orc1 AAA+ domain with other AAA+ proteins in E. coli could be stimulated in certain orc1 mutants.


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

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

Construction of ORC-overproducing plasmids. All the pHK plasmids used in this study are derivatives of ver. 3–5. See text for details.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 2: Construction of ORC-overproducing plasmids. All the pHK plasmids used in this study are derivatives of ver. 3–5. See text for details.
Mentions: To overproduce S. cerevisiae ORC in mammalian cells, we modified the ver. 3–5 vector for transfection, which was originally developed to overexpress proteins that are not overexpressed well in bacterial or insect cells (Figure 2; Uno and Masai, 2011; Uno et al., 2012). Ver. 3–5 is a shuttle vector carrying PEF−1α, a Kozak sequence, a His tag, a multiple cloning site, and an HA tag. Ver. 3–5 also bears an SV40 origin, which could help to maintain the vector episomally in mammalian cells expressing the large T antigen, such as 293T cells. To maintain the plasmid in Escherichia coli, ver. 3–5 also bears bla and ColE1 ori. We first cloned one of the ORC1/2/3/4/5/6 genes into ver. 3–5 so that the N-terminal His tag and C-terminal HA tag of the vector were eliminated, yielding pHK106 through to pHK111. By site-directed mutagenesis, a hexahistidine sequence with a linker was appended just before the stop codon of ORC1, yielding pHK118. This tag, consisting of 13 amino acids, is shorter than a CBP-TEV tag previously used for Orc1 tagging (Frigola et al., 2013). Addition of a short tag, such as His12 or His-Strep II, to the C-terminus of Orc1 does not affect Orc1 function in vivo (Kawakami et al., 2015). We noticed that introduction of certain orc1 mutations into pHK118 by site-directed mutagenesis was unsuccessful. Because the same mutation could be introduced into another ORC1 plasmid under the control of the native ORC1 promoter using the same mutagenic primers (Kawakami et al., 2015), one plausible idea is that leaky expression of Orc1 from pHK118 may be extremely toxic in E. coli cells only when a certain orc1 mutation is introduced. Because Orc1 solely binds to the ARS sequence via a domain termed EOS (Kawakami et al., 2015), similar binding to a similar sequence in the E. coli genome may be affected by the mutation and interfere with a certain cellular process in vivo. Alternatively, adverse interactions of the Orc1 AAA+ domain with other AAA+ proteins in E. coli could be stimulated in certain orc1 mutants.

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