Large-scale purification and crystallization of the endoribonuclease XendoU: troubleshooting with His-tagged proteins

Acta Crystallographica. Section F, Structural Biology and Crystallization Communications
Fabiana RenziElisa Caffarelli

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.

References

Apr 20, 2000·Acta Crystallographica. Section D, Biological Crystallography·V R SamyginaA N Popov
May 22, 2002·Biochimica Et Biophysica Acta·Fabiana RenziElisa Caffarelli
Jun 18, 2002·Current Opinion in Cell Biology·Witold Filipowicz, Vanda Pogacić
Apr 30, 2003·Journal of Structural Biology·W William Wilson
Aug 12, 2004·Proceedings of the National Academy of Sciences of the United States of America·Konstantin A IvanovJohn Ziebuhr
Mar 10, 2005·The Journal of Biological Chemistry·Ubaldo GioiaElisa Caffarelli

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Citations

Feb 15, 2011·Biotechnology Letters·Wen-Hui K Kuo, Howard A Chase
Oct 22, 2008·The Journal of Biological Chemistry·Pietro LaneveElisa Caffarelli
Aug 30, 2011·Protein Expression and Purification·David S Waugh
Jan 1, 2013·Biomolecules·János András MótyánJózsef Tőzsér
Jul 6, 2017·Scientific Reports·Thomas FolliardLynn J Rothschild
Aug 15, 2006·Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences·Mouna Amor-MahjoubMoncef Ladjimi

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Methods Mentioned

BETA
gel filtration
X-ray

Software Mentioned

MOSFLM
CCP
XendoU
SCALA
HKL

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