Large-scale purification and crystallization of the endoribonuclease XendoU: troubleshooting with His-tagged proteins
- Dipartimento di Scienze Biochimiche, University of Rome ‘La Sapienza’, Piazzale Aldo Moro 5, 00185 Roma (Italy)
- Istituto di Biologia e Patologia Molecolari CNR, University of Rome ‘La Sapienza’, Piazzale Aldo Moro 5, 00185 Roma (Italy)
- Istituto Pasteur-Fondazione Cenci Bolognetti, University of Rome ‘La Sapienza’, Piazzale Aldo Moro 5, 00185 Roma (Italy)
Recombinant His-tagged XendoU, a eukaryotic endoribonuclease, appeared to aggregate in the presence of divalent cations. Monodisperse protein which yielded crystals diffracting to 2.2 Å was obtained by addition of EDTA. 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{sub 1}21, diffracting to low resolution, and monoclinic C2, diffracting to higher resolution.
- OSTI ID:
- 22356310
- Journal Information:
- Acta Crystallographica. Section F, Vol. 62, Issue Pt 3; Other Information: PMCID: PMC2197201; PMID: 16511328; PUBLISHER-ID: za5130; OAI: oai:pubmedcentral.nih.gov:2197201; Copyright (c) International Union of Crystallography 2006; Country of input: International Atomic Energy Agency (IAEA); ISSN 1744-3091
- Country of Publication:
- United Kingdom
- Language:
- English
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