Purification, crystallization and preliminary X-ray diffraction analysis of Cif, a virulence factor secreted by Pseudomonas aeruginosa

Abstract

The opportunistic pathogen Pseudomonas aeruginosa secretes a protein that triggers the accelerated degradation of the cystic fibrosis transmembrane conductance regulator (CFTR) in airway epithelial cells. This protein, which is known as the CFTR inhibitory factor (Cif), acts as a virulence factor and may facilitate airway colonization by P. aeruginosa. Based on sequence similarity Cif appears to be an epoxide hydrolase (EH), but it lacks several of the conserved features found in the active sites of canonical members of the EH family. Here, the crystallization of purified recombinant Cif by vapor diffusion is reported. The crystals formed in space group C2, with unit-cell parameters a = 167.4, b = 83.6, c = 88.3 A, beta = 100.6 degrees . The crystals diffracted to 2.39 A resolution on a rotating-anode source. Based on the calculated Matthews coefficient (2.2 A(3) Da(-1)), it appears that the asymmetric unit contains four molecules.

Figure 1

Crystallization of Cif. (a) A representative crystal of Cif obtained by hanging-drop vapor diffusion. The well solution contained 15%(w/v) PEG 8000, 125 mM CaCl₂ and 100 mM sodium acetate pH 5. (b) An SDS–PAGE gel of a washed Cif crystal, visualized by silver staining. A sample of mother liquor from the same drop was washed in an identical manner and gave no visible band (data not shown).