Expression, purification, characterization and crystallization of a recombinant human cytosolic β-glucosidase produced in insect cells

Human cytosolic β-glucosidase is a monomeric enzyme that hydrolyzes various β-d-glycosides and its real physiological role remains unclear. Here, we describe the production of this enzyme in Sf9 cells with a N-terminal 6× His tag. The production yield of the recombinant protein was in the 10 to 30 mg/l range. The protein was purified to homogeneity using two chromatographic steps, taking advantage of the 6× His tag in the first step, then using the physical and chemical properties of the protein for ionic exchange. Gel filtration analysis revealed that the protein is monomeric as expected. The kinetic parameters for 4-methylumbelliferyl β-l-glucopyranoside, VM and KM, were measured (KM = 32 μM and VM = 157 μmol/h/mg at pH 7.0) and found similar to those reported for either the natural isolated enzyme or the recombinant protein expressed in COS7 cells (KM of 60–70 μM and 40 μM, respectively). Protein crystals were obtained and are now under structural investigations. In summary, we set up a heterologous expression system in Sf9 insect cells allowing the expression and production of large amounts of a pure active human protein, suitable for crystallographic studies.