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Updated: Aug 9, 2026
Overexpressing and Purifying a Toxic Nuclease from Escherichia coli
Published on: August 29, 2025
Expression, purification and crystallization of a birnavirus-encoded protease, VP4, from blotched snakehead virus
Jaeyong Lee1, Anat R Feldman, Bernard Delmas
1Department of Molecular Biology and Biochemistry, Simon Fraser University, South Science Building, 8888 University Drive, Burnaby, British Columbia, V5A 1S6, Canada.
Abstract:
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.
