Cloning, expression, purification, characterization, crystallization and X-ray crystallographic analysis of recombinant Derf21 (rDerf21) from Dermatophagoides farinae

Abstract

Dermatophagoides farinae is one of the major house dust mite (HDM) species that cause allergic diseases. N-terminally His-tagged recombinant Derf21 (rDerf21), a group 21 allergen, with the signal peptide truncated was successfully overexpressed in an Escherichia coli expression system. The purified rDerf21 protein was initially crystallized using the sitting-drop vapour-diffusion method. Well diffracting protein crystals were obtained after optimization of the crystallization conditions using the hanging-drop vapour-diffusion method with a reservoir solution consisting of 0.19M Tris-HCl pH 8.0, 32% PEG 400 at 293K. X-ray diffraction data were collected to 1.49Å resolution using an in-house X-ray source. The crystal belonged to the C-centered monoclinic space group C2, with unit-cell parameters a = 123.46, b = 27.71, c = 90.25Å, ? = 125.84°. The calculated Matthews coefficient (V M) of 2.06Å3Da-1 suggests that there are two molecules per asymmetric unit, with a solvent content of 40.3%. Despite sharing high sequence identity with Blot5 (45%) and Blot21 (41%), both of which were determined to be monomeric in solution, size-exclusion chromatography, static light scattering and self-rotation function analysis indicate that rDerf21 is likely to be a dimeric protein. © 2015 International Union of Crystallography.