Expression, purification and crystallization of a PCI domain from the COP9 signalosome subunit 7 (CSN7)

Abstract

A core fragment of Arabidopsis thaliana COP9 signalosome (CSN) subunit 7 was expressed in Escherichia coli. The protein was purified to homogeneity and screened for crystallization. Crystallization conditions were refined using the sitting-drop vapour-diffusion method. Crystals were obtained using polyethylene glycol 8000 as a precipitant and have a thick rod-like morphology. Their crystallographic symmetry is orthorhombic, space group C222(1), with unit-cell parameters a = 57.2, b = 86.2, c = 72.6 A and a diffraction limit of 2.06 A.

Figure 1

SDS–PAGE of the AtCSN7 core purification steps. Lane 1, material loaded onto Ni²⁺-chelate column; lane 2, sample of unbound material; lane 3, elution from the Ni²⁺ -chelate column obtained with 50 mM imidazole; lane 4, protein after overnight TEV protease digestion; lane 5, sample of unbound material from second Ni²⁺-chelate column; lane 6, sample of material loaded onto Superdex 200 gel-filtration column; lane 7, relevant gel-filtration fractions. MW markers (extreme left) are from top to bottom: 97, 66, 45, 31, 21, 14 kDa. The gel was Coomassie-stained.

Figure 2

(a) A cluster of rod-shaped crystals of the AtCSN7 core as obtained in the initial screen. The cystals are approximately 0.05–0.1 mm in length. (b) Rod-shaped crystals of the AtCSN7 core obtained after lowering the magnesium acetate concentration. The crystals are approximately 0.15–0.3 mm in the longest dimension. The crystallization conditions for these crystals were a protein concentration of 10 mg ml⁻¹ with a reservoir comprising 23%(w/v) PEG 8000, 0.1 M sodium cacodylate pH 7.2 and 0.08 M magnesium acetate.

Figure 3

Oscillation image (1°) of AtCSN7 core protein crystals. The crystal-to-detector distance was 180 mm. Diffraction data were observed to 2.06 Å. Data were collected and processed to a d _min of 2.1 Å. Crystallization conditions were the same as for Fig. 2 ▶(b).