Expression, purification and crystallization of the SARS-CoV macro domain.
Macro domains or X domains are found as modules of multidomain proteins, but can also constitute a protein on their own. Recently, biochemical and structural studies of cellular macro domains have been performed, showing that they are active as ADP-ribose-1''-phosphatases. Macro domains are also present in a number of positive-stranded RNA viruses, but their precise function in viral replication is still unknown. The major human pathogen severe acute respiratory syndrome coronavirus (SARS-CoV) encodes 16 non-structural proteins (nsps), one of which (nsp3) encompasses a macro domain. The SARS-CoV nsp3 gene region corresponding to amino acids 182-355 has been cloned, expressed in Escherichia coli, purified and crystallized. The crystals belong to space group P2(1), with unit-cell parameters a = 37.5, b = 55.6, c = 108.9 angstroms, beta = 91.4 degrees, and the asymmetric unit contains either two or three molecules. Both native and selenomethionine-labelled crystals diffract to 1.8 angstroms.
Centre National de la Recherche Scientifique and Universités d'Aix-Marseille I et II, UMR 6098, Architecture et Fonction des Macromolécules Biologiques, UMR 6098-Case 932, 163 Avenue de Luminy, 13288 Marseille CEDEX 9, France.
Hélène
H
Malet
Karen
K
Dalle
Nicolas
N
Brémond
Fabienne
F
Tocque
Stéphanie
S
Blangy
Valérie
V
Campanacci
Bruno
B
Coutard
Sacha
S
Grisel
Julie
J
Lichière
Violaine
V
Lantez
Christian
C
Cambillau
Bruno
B
Canard
Marie Pierre
MP
Egloff
D003001:Cloning, Molecular; D003460:Crystallization; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D012324:RNA-Dependent RNA Polymerase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D045473:Severe acute respiratory syndrome-related coronavirus / Q000201:enzymology*; D017361:Viral Nonstructural Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D014961:X-Ray Diffraction
D010446:Peptide Fragments; D011994:Recombinant Proteins; D017361:Viral Nonstructural Proteins; D014764:Viral Proteins; C510959:Nsp3 protein, SARS-CoV; D012324:RNA-Dependent RNA Polymerase