title: Expression, high-pressure refolding, purification, crystallization and preliminary X-ray analysis of a novel single-strand-specific 3'-5' exonuclease PhoExo I from Pyrococcus horikoshii OT3.
abstract: PhoExo I is a single-strand-specific 3'-5' exonuclease from Pyrococcus horikoshii OT3 and is thought to be involved in a Thermococcales-specific DNA-repair pathway. The recombinant PhoExo I protein was produced as inclusion bodies in Escherichia coli cells. Solubilization of the inclusion bodies was performed by the high-pressure refolding method and highly purified protein was subjected to crystallization by the sitting-drop vapour-diffusion method at 20°C. A crystal of PhoExo I was obtained in a reservoir solution consisting of 0.1 M Tris-HCl pH 8.9, 27% PEG 6000 and diffracted X-rays to 1.52 Å resolution. The crystal of PhoExo I belonged to space group H32, with unit-cell parameters a = b = 112.07, c = 202.28 Å. The crystal contained two PhoExo I molecules in the asymmetric unit.
authors.affiliation: Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
authors.forename: Ken-ichi
authors.initials: K
authors.lastname: Miyazono
authors.affiliation: Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
authors.forename: Kanae
authors.initials: K
authors.lastname: Tsutsumi
authors.affiliation: Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, and Faculty of Agriculture, Kyushu University, Fukuoka, Japan.
authors.forename: Yoshizumi
authors.initials: Y
authors.lastname: Ishino
authors.affiliation: Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
authors.forename: Masaru
authors.initials: M
authors.lastname: Tanokura
mesh_terms: D003460:Crystallization; D004591:Electrophoresis, Polyacrylamide Gel; D005092:Exonucleases / Q000378:metabolism*; D011312:Pressure; D043902:Pyrococcus horikoshii / Q000201:enzymology*
chemical_list: D005092:Exonucleases
keywords: DNA repair; PhoExo I; Pyrococcus horikoshii; high-pressure refolding; nuclease
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