Expression, purification and crystallization of a birnavirus-encoded protease, VP4, from blotched snakehead virus (BSNV).
Blotched snakehead virus (BSNV) is a member of the Birnaviridae family that requires a virally encoded protease known as VP4 in order to process its polyprotein into viral capsid protein precursors (pVP2 and VP3). VP4 belongs to a family of serine proteases that utilize a serine/lysine catalytic dyad mechanism. A mutant construct of VP4 with a short C-terminal truncation was overexpressed in Escherichia coli and purified to homogeneity for crystallization. Using the sitting-drop vapour-diffusion method at room temperature, protein crystals with two distinct morphologies were observed. Cubic crystals grown in PEG 2000 MME and magnesium acetate at pH 8.5 belong to space group I23, with unit-cell parameters a = b = c = 143.8 angstroms. Trigonal crystals grown in ammonium sulfate and glycerol at pH 8.5 belong to space group P321/P312, with unit-cell parameters a = b = 158.2, c = 126.4 angstroms.
Department of Molecular Biology and Biochemistry, Simon Fraser University, South Science Building, 8888 University Drive, Burnaby, British Columbia, V5A 1S6, Canada.
Jaeyong
J
Lee
Anat R
AR
Feldman
Bernard
B
Delmas
Mark
M
Paetzel
D001483:Base Sequence; D018059:Birnaviridae / Q000201:enzymology*; D018360:Crystallography, X-Ray; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D012542:Scattering, Radiation; D012697:Serine Endopeptidases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification
D017931:DNA Primers; D012697:Serine Endopeptidases; C414992:VP4 protease, birnavirus