Overexpression, purification and crystallization of the two C-terminal domains of the bifunctional cellulase ctCel9D-Cel44A from Clostridium thermocellum

Abstract

Clostridium thermocellum produces a highly organized multi-enzyme complex of cellulases and hemicellulases for the hydrolysis of plant cell-wall polysaccharides, which is termed the cellulosome. The bifunctional multi-modular cellulase ctCel9D-Cel44A is one of the largest components of the C. thermocellum cellulosome. The enzyme contains two internal catalytic domains belonging to glycoside hydrolase families 9 and 44. The C-terminus of this cellulase, comprising a polycystic kidney-disease module (PKD) and a carbohydrate-binding module (CBM44), has been crystallized. The crystals belong to the tetragonal space group P4(3)2(1)2, containing a single molecule in the asymmetric unit. Native and seleno-L-methionine-derivative crystals diffracted to 2.1 and 2.8 A, respectively.

Figure 1

A Coomassie Brilliant Blue-stained 14% SDS–PAGE gel evaluation of protein purity during purification. Lane M, molecular-weight markers (kDa); lane 1, cell debris after sonication; lane 2, cell-free extract after sonication; lane 3, eluate from the column after sample loading; lane 4, purified PKD-CBM44 protein after Ni-affinity column chromatography.

Figure 2

Crystals of PKD-CBM44 protein obtained by hanging-drop vapour diffusion in the presence of 0.2 M CaCl₂, 0.1 M sodium acetate pH 4.5 and 30% ethanol with 6% glycerol as additive. The largest crystals are approximately 0.1 × 0.1 × 0.2 mm in size.