Expression, purification, characterization and crystallization of a recombinant human cytosolic beta-glucosidase produced in insect cells.
Human cytosolic beta-glucosidase is a monomeric enzyme that hydrolyzes various beta-d-glycosides and its real physiological role remains unclear. Here, we describe the production of this enzyme in Sf9 cells with a N-terminal 6x 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 6x 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 beta-L-glucopyranoside, VM and KM, were measured (KM=32 microM and VM=157 micromol/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 microM and 40 microM, 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.
Fleury
Damien
D
Prostrakan, Protein Production and Crystallization, 102 route de Noisy, 93230 Romainville, France. damien.fleury@prostrakan.com
Domaingue
Priscilla
P
Gillard
Corinne
C
Touitou
Robert
R
Mollat
Patrick
P
D000818:Animals; D002460:Cell Line; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D006801:Humans; D007313:Insecta; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012995:Solubility; D018411:Spodoptera; D014162:Transfection; D001617:beta-Glucosidase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D011994:Recombinant Proteins; D001617:beta-Glucosidase