Expression, refolding, crystallization and preliminary X-ray analysis of Pseudomonas aeruginosa AlgE

Abstract

AlgE is an outer membrane protein present in alginate-producing (mucoid) Pseudomonas aeruginosa. AlgE has been overexpressed in insoluble inclusion bodies, purified under denaturing conditions and refolded in a buffer containing decyl beta-D-maltopyranoside. Purified refolded AlgE was detergent-exchanged into n-octyl tetraoxyethylene and diffraction-quality crystals were grown using the hanging-drop vapor-diffusion method. The crystals grew as small hexagons with unit-cell parameters a = 98.8, b = 156.8, c = 90.4 A, alpha = beta = gamma = 90.0 degrees . The crystals exhibited the symmetry of space group C222 and diffracted to a minimum d-spacing of 3.0 A. On the basis of the Matthews coefficient (V(M) = 3.28 A(3) Da(-1)), one molecule is estimated to be present in the asymmetric unit.

Figure 1

SDS–PAGE analysis of AlgE refolding and purification. Samples loaded on the gel were not boiled unless otherwise indicated. (a) Lane M, molecular-weight markers (kDa); lane 1, purified urea-solubilized AlgE after Ni-affinity chromatography; lane 2, AlgE after refolding in buffer containing decyl β-d-maltopyranoside; lane 3, refolded AlgE after digestion with 500:1(w:w) trypsin overnight at room temperature (295 K). (b) Lane M, molecular-weight markers (kDa); lane 1, refolded AlgE after size-exclusion chromatography; lane 2, as lane 1 except boiled for 5 min prior to SDS–PAGE analysis. Unfolded and folded forms of AlgE are designated U and F, respectively.

Figure 2

Crystals of refolded AlgE. The crystal dimensions are approximately 100 × 50 × 25 µm.