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

Abstract

Plant β-galactosidases play important roles in carbohydrate-reserve mobilization, cell-wall expansion and degradation, and turnover of signalling molecules during ripening. Tomato β-galactosidase 4 (TBG4) not only has β-galactosidase activity but also has exo-β-(1,4)-galactanase activity, and prefers β-(1,4)-galactans longer than pentamers as its substrates; most other β-galactosidases only have the former activity. Recombinant TBG4 protein expressed in the yeastPichia pastoriswas crystallized by the sitting-drop vapour-diffusion method using PEG 10 000 as a precipitant. The crystals belonged to the orthorhombic space groupP212121, with unit-parametersa= 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 Å3 Da−1), with a solvent content of 45%.