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

Acta Crystallogr Sect F Struct Biol Cryst Commun. 2006 Mar 1;62(Pt 3):298-301. doi: 10.1107/S1744309106006373. Epub 2006 Feb 28.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Crystallization / methods
  • Edetic Acid / chemistry
  • Endoribonucleases / chemistry*
  • Endoribonucleases / isolation & purification*
  • Escherichia coli / metabolism
  • Histidine / genetics*
  • Oligopeptides / genetics*
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / isolation & purification
  • Xenopus Proteins / chemistry*
  • Xenopus Proteins / isolation & purification*
  • Xenopus laevis

Substances

  • His-His-His-His-His-His
  • Oligopeptides
  • Recombinant Fusion Proteins
  • Xenopus Proteins
  • Histidine
  • Edetic Acid
  • Endoribonucleases
  • ENDOU protein, Xenopus