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Acta Crystallogr Sect F Struct Biol Cryst Commun
2006 Mar 01;62Pt 3:298-301. doi: 10.1107/S1744309106006373.
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Large-scale purification and crystallization of the endoribonuclease XendoU: troubleshooting with His-tagged proteins.
Renzi F, Panetta G, Vallone B, Brunori M, Arceci M, Bozzoni I, Laneve P, Caffarelli E.
???displayArticle.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.
Figure 1. Effect of chelating agents in the solubilization of aggregated His-tagged protein. HPLC gel-filtration analysis shows that 250 mM (red) and 100 mM (blue) imidazole yield monodisperse His-tagged protein. 20 mM (light green), 10 mM (dark green) and 5 mM (purple) EDTA also improve monodispersity in a concentration-dependent fashion. Addition of 20 mM Mn2+ salt with and without EDTA (brown and pink) causes the protein to aggregate again. Elution profiles were analyzed using the CSW program.
Figure 2. (a) Trigonal crystals in 40% (NH4)2SO4, 0.2 M sodium citrate pH 6, 17 mg ml−1 protein, 50 mM UMP at 293 K and (b) their diffraction image with an enlargement. (c) Monoclinic crystals in 40% NH4SO4, 0.2 M sodium phosphate pH 5.5, 17 mg ml−1 protein, 50 mM UMP at 293 K improved by seeding from aggregated (top) to single crystals (bottom) and (d) their diffraction image with an enlargement.
Caffarelli,
In vitro study of processing of the intron-encoded U16 small nucleolar RNA in Xenopus laevis.
1994, Pubmed,
Xenbase
Caffarelli,
In vitro study of processing of the intron-encoded U16 small nucleolar RNA in Xenopus laevis.
1994,
Pubmed
,
Xenbase Collaborative Computational Project, Number 4,
The CCP4 suite: programs for protein crystallography.
1994,
Pubmed Filipowicz,
Biogenesis of small nucleolar ribonucleoproteins.
2002,
Pubmed Fragapane,
A novel small nucleolar RNA (U16) is encoded inside a ribosomal protein intron and originates by processing of the pre-mRNA.
1993,
Pubmed
,
Xenbase Gioia,
Functional characterization of XendoU, the endoribonuclease involved in small nucleolar RNA biosynthesis.
2005,
Pubmed
,
Xenbase Ivanov,
Major genetic marker of nidoviruses encodes a replicative endoribonuclease.
2004,
Pubmed Laneve,
Purification, cloning, and characterization of XendoU, a novel endoribonuclease involved in processing of intron-encoded small nucleolar RNAs in Xenopus laevis.
2003,
Pubmed
,
Xenbase Renzi,
Characterization of the sequences encoding for Xenopus laevis box C/D snoRNP Nop56 protein.
2002,
Pubmed
,
Xenbase Samygina,
Improving the X-ray resolution by reversible flash-cooling combined with concentration screening, as exemplified with PPase.
2000,
Pubmed Snijder,
Unique and conserved features of genome and proteome of SARS-coronavirus, an early split-off from the coronavirus group 2 lineage.
2003,
Pubmed Wilson,
Light scattering as a diagnostic for protein crystal growth--a practical approach.
2003,
Pubmed
