title: Large-scale purification and crystallization of the endoribonuclease XendoU: troubleshooting with His-tagged proteins.
abstract: XendoU is the first endoribonuclease described in higher eukaryotes as being involved in the endonucleolytic processing of intron-encoded small nucleolar RNAs. It is conserved among eukaryotes and its viral homologue is essential in SARS replication and transcription. The large-scale purification and crystallization of recombinant XendoU are reported. The tendency of the recombinant enzyme to aggregate could be reversed upon the addition of chelating agents (EDTA, imidazole): aggregation is a potential drawback when purifying and crystallizing His-tagged proteins, which are widely used, especially in high-throughput structural studies. Purified monodisperse XendoU crystallized in two different space groups: trigonal P3(1)21, diffracting to low resolution, and monoclinic C2, diffracting to higher resolution.
authors.affiliation: Dipartimento di Scienze Biochimiche, University of Rome La Sapienza, Piazzale Aldo Moro 5, 00185 Rome, Italy.
authors.forename: Fabiana
authors.initials: F
authors.lastname: Renzi
authors.forename: Gianna
authors.initials: G
authors.lastname: Panetta
authors.forename: Beatrice
authors.initials: B
authors.lastname: Vallone
authors.forename: Maurizio
authors.initials: M
authors.lastname: Brunori
authors.forename: Massimo
authors.initials: M
authors.lastname: Arceci
authors.forename: Irene
authors.initials: I
authors.lastname: Bozzoni
authors.forename: Pietro
authors.initials: P
authors.lastname: Laneve
authors.forename: Elisa
authors.initials: E
authors.lastname: Caffarelli
mesh_terms: D000818:Animals; D003460:Crystallization / Q000379:methods; D004492:Edetic Acid / Q000737:chemistry; D004722:Endoribonucleases / Q000737:chemistry* / Q000302:isolation & purification*; D004926:Escherichia coli / Q000378:metabolism; D006639:Histidine / Q000235:genetics*; D009842:Oligopeptides / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification; D029867:Xenopus Proteins / Q000737:chemistry* / Q000302:isolation & purification*; D014982:Xenopus laevis
chemical_list: C471213:His-His-His-His-His-His; D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; D029867:Xenopus Proteins; D006639:Histidine; D004492:Edetic Acid; D004722:Endoribonucleases; C475059:ENDOU protein, Xenopus