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

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

SUBMITTER: Kawakami H 

PROVIDER: S-EPMC4834435 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature