Expression, purification, crystallization and preliminary X-ray crystallographic analysis of tomato ß-galactosidase 4.

ABSTRACT

Plant ß-galactosidases play important roles in carbohydrate-reserve mobilization, cell-wall expansion and degradation, and turnover of signalling molecules during ripening. Tomato ß-galactosidase 4 (TBG4) not only has ß-galactosidase activity but also has exo-ß-(1,4)-galactanase activity, and prefers ß-(1,4)-galactans longer than pentamers as its substrates; most other ß-galactosidases only have the former activity. Recombinant TBG4 protein expressed in the yeast Pichia pastoris was crystallized by the sitting-drop vapour-diffusion method using PEG 10,000 as a precipitant. The crystals belonged to the orthorhombic space group P212121, with unit-parameters a = 92.82, b = 96.30, c = 159.26 Å, and diffracted to 1.65 Å resolution. Calculation of the Matthews coefficient suggested the presence of two monomers per asymmetric unit (VM = 2.2 Å(3) Da(-1)), with a solvent content of 45%.