Crystallization and preliminary X-ray data analysis of a DJ-1 homologue from Arabidopsis thaliana (AtDJ-1D)

Abstract

A DJ-1 homologue protein from Arabidopsis thaliana (AtDJ-1D) belongs to the DJ-1/ThiJ/Pfpl superfamily and contains two tandem arrays of DJ-1-like sequences, but no structural information is available to date for this protein. AtDJ-1D was expressed in Escherichia coli, purified and crystallized for structural analysis. A crystal of AtDJ-1D was obtained by the hanging-drop vapour-diffusion method using 0.22 M NaCl, 0.1 M bis-tris pH 6.5, 21% polyethylene glycol 3350. AtDJ-1D crystals belonged to the monoclinic space group P2(1), with unit-cell parameters a = 56.78, b = 75.21, c = 141.68 Å, β = 96.87°, and contained a trimer in the asymmetric unit. Diffraction data were collected to 2.05 Å resolution. The structure of AtDJ-1D has been determined using the multiple-wavelength anomalous dispersion (MAD) method.

Figure 1

(a) SDS–PAGE analysis of AtDJ-1D at different steps of the purification process. Lane 1, marker (labelled in kDa); lane 2, total cell lysate; lane 3, supernatant after cell lysis; lane 4, pellet after cell lysis; lane 5, proteins from an Ni–NTA affinity column; lane 6, proteins separated after anion-exchange chromatography; lane 7, proteins separated after Superdex 200 chromatography (8.5 µg protein used). (b) Superdex 200 size-exclusion chromatography of AtDJ-1D. A single peak corresponding to a monomer was observed. The scale at the bottom indicates the elution volume. Inset, semi log plot of the molecular mass of the standard proteins used versus their logK _d values.

Figure 2

Crystal of AtDJ-1D obtained by the hanging-drop method.