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.
Highlights
Purification of mutant proteins from overproducing cells constituted a milestone in biochemistry to analyze proteins of interest
origin recognition complex (ORC) binds to eukaryotic chromosomal replication origins in an ATP-dependent manner to recruit Cdc6, Cdt1, and the MCM2-7 helicase core onto double-stranded DNA (Boos et al, 2012; Bell and Kaguni, 2013; Yardimci and Walter, 2014; Tognetti et al, 2015)
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)
Summary
Purification of mutant proteins from overproducing cells constituted a milestone in biochemistry to analyze proteins of interest. One approach is to use insect cells cotransfected with three types of baculoviruses carrying two of the six ORC subunits (Bell et al, 1995; Fujita et al, 1998; Sun et al, 2012, 2013; Samel et al, 2014) Another approach is to construct a yeast strain with inducible promoters (Remus et al, 2009; Hizume et al, 2013). An improved method was developed to overcome part of these problems in purification of ORC containing a site- mutated Orc subunit (Frigola et al, 2013; Coster et al, 2014) In this method, CBP (calmodulin-binding peptide)-tagged Orc, mutant Orc, and intact Orc2/3/5/6 were co-expressed in a yeast strain in which endogenous wild-type Orc is Flagtagged. Purified wild-type and mutant ORCs using this system showed expected biochemical activities; the rapid acquisition of mutant ORCs using this highly versatile system will boost systematic biochemical dissection of ORC and can be even applied to purify protein complexes other than ORC
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