title: Large-scale production of recombinant Saw1 in Escherichia coli.
abstract: Saccharomyces cerevisiae Saw1 is an essential gene in single-strand annealing - the DNA repair pathway that repairs double-strand breaks when they occur between homologous repeats. Saw1 interacts with the structure-specific nuclease Rad1-Rad10 and this results in the recruitment of this nuclease to 3' non-homologous DNA tailed recombination intermediates. Saw1 is unstable in the absence of the Rad1-Rad10 nuclease and, hence, it has been difficult to study its specific function in vitro. In the present work, we present the combination of dynamic light scattering and differential scanning fluorimetry techniques to optimize the stability and homogeneity of recombinant Saw1. The protein expression and purification conditions identified in this study allow for higher recovery of soluble Saw1 and enable the biochemical characterization of the protein.
authors.affiliation: Department of Biochemistry and Biomedical Sciences, McMaster University, ON, Canada.
authors.forename: Margarita
authors.initials: M
authors.lastname: Rashev
authors.affiliation: Department of Biochemistry, Jacobs School of Medicine and Biomedical Sciences, SUNY at Buffalo, NY, USA.
authors.forename: Jennifer A
authors.initials: JA
authors.lastname: Surtees
authors.affiliation: Department of Biochemistry and Biomedical Sciences, McMaster University, ON, Canada. Electronic address: guarnea@mcmaster.ca.
authors.forename: Alba
authors.initials: A
authors.lastname: Guarné
mesh_terms: D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
chemical_list: D004268:DNA-Binding Proteins; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; C579349:Saw1 protein, S cerevisiae
keywords: Differential scanning fluorimetry; Protein stability; Recombination intermediates; SYPRO Orange; Single-strand annealing