Expression, purification, crystallization and preliminary X-ray analysis of rice (Oryza sativa L.) Os4BGlu12 beta-glucosidase

Abstract

Rice (Oryza sativa L.) Os4BGlu12, a glycoside hydrolase family 1 beta-glucosidase (EC 3.2.1.21), was expressed as a fusion protein with an N-terminal thioredoxin/His(6) tag in Escherichia coli strain Origami B (DE3) and purified with subsequent removal of the N-terminal tag. Native Os4BGlu12 and its complex with 2,4-dinitrophenyl-2-deoxy-2-fluoro-beta-D-glucopyranoside (DNP2FG) were crystallized using 19% polyethylene glycol (3350 or 2000, respectively) in 0.1 M Tris-HCl pH 8.5, 0.16 M NaCl at 288 K. Diffraction data sets for the apo and inhibitor-bound forms were collected to 2.50 and 2.45 A resolution, respectively. The space group and the unit-cell parameters of the crystal indicated the presence of two molecules per asymmetric unit, with a solvent content of 50%. The structure of Os4BGlu12 was successfully solved in space group P4(3)2(1)2 by molecular replacement using the white clover cyanogenic beta-glucosidase structure (PDB code 1cbg) as a search model.

Figure 1

12% SDS–PAGE of Os4BGlu12 protein from the purification steps. Lane M, protein markers (kDa); lane 1, soluble protein extract of E. coli cells after Os4BGlu12 expression; lane 2, the N-terminal thioredoxin/His₆-tagged Os4BGlu12 fusion protein after the first Co²⁺-IMAC column; lane 3, enterokinase digest of the fusion protein; lane 4, pure Os4BGlu12 after removal of the cleaved thioredoxin/His₆ tag by the second Co²⁺-IMAC column.

Figure 2

Single crystals of (a) native Os4BGlu12 and (b) Os4BGlu12 complexed with DNP2FG obtained after optimization, with maximum dimensions of 120 × 25 × 20 and 125 × 25 × 25 µm, respectively.