Open AccessLarge‐scale purification and crystallization of the endoribonuclease XendoU: troubleshooting with His‐tagged proteins
Author(s) -
Renzi Fabiana,
Panetta Gianna,
Vallone Beatrice,
Brunori Maurizio,
Arceci Massimo,
Bozzoni Irene,
Laneve Pietro,
Caffarelli Elisa
Publication year - 2006
Publication title -
acta crystallographica section f
Language(s) - English
Resource type - Journals
ISSN - 1744-3091
DOI - 10.1107/s1744309106006373
Subject(s) - endoribonuclease , recombinant dna , crystallization , biology , chemistry , computational biology , microbiology and biotechnology , biochemistry , rna , gene , rnase p , organic chemistry
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 P 3 1 21, diffracting to low resolution, and monoclinic C 2, diffracting to higher resolution.