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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, 0.2â M sodium citrate pH 6, 17â mgâ mlâ1 protein, 50â mM UMP at 293â K and (b) their diffraction image with an enlargement. (c) Monoclinic crystals in 40% NH4SO4, 0.2â M sodium phosphate pH 5.5, 17â mgâ mlâ1 protein, 50â mM UMP at 293â K improved by seeding from aggregated (top) to single crystals (bottom) and (d) their diffraction image with an enlargement.
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