Large-scale purification and crystallization of the endoribonuclease XendoU: troubleshooting with His-tagged proteins.
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.
Renzi
Fabiana
F
Dipartimento di Scienze Biochimiche, University of Rome La Sapienza, Piazzale Aldo Moro 5, 00185 Rome, Italy.
Panetta
Gianna
G
Vallone
Beatrice
B
Brunori
Maurizio
M
Arceci
Massimo
M
Bozzoni
Irene
I
Laneve
Pietro
P
Caffarelli
Elisa
E
D000818:Animals; D003460:Crystallization / Q000379:methods; D004492:Edetic Acid / Q000737:chemistry; D004722:Endoribonucleases / Q000737:chemistry* / Q000302:isolation & purification*; D004926:Escherichia coli / Q000378:metabolism; D006639:Histidine / Q000235:genetics*; D009842:Oligopeptides / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification; D029867:Xenopus Proteins / Q000737:chemistry* / Q000302:isolation & purification*; D014982:Xenopus laevis
C471213:His-His-His-His-His-His; D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; D029867:Xenopus Proteins; D006639:Histidine; D004492:Edetic Acid; D004722:Endoribonucleases; C475059:ENDOU protein, Xenopus