Purification, crystallization and preliminary X-ray analysis of rice BGlu1 beta-glucosidase with and without 2-deoxy-2-fluoro-beta-D-glucoside

Abstract

Rice (Oryza sativa) BGlu1 beta-glucosidase was expressed in Escherichia coli with N-terminal thioredoxin and hexahistidine tags and purified by immobilized metal-affinity chromatography (IMAC). After removal of the N-terminal tags, cation-exchange and S-200 gel-filtration chromatography yielded a 50 kDa BGlu1 with >95% purity. The free enzyme and a complex with 2,4-dinitrophenyl-2-deoxy-2-fluoro-beta-D-glucopyranoside inhibitor were crystallized by microbatch and hanging-drop vapour diffusion. Small tetragonal crystals of BGlu1 with and without inhibitor grew in 18%(w/v) PEG 8000 with 0.1 M sodium cacodylate pH 6.5 and 0.2 M zinc acetate. Crystals of BGlu1 with inhibitor were streak-seeded into 23%(w/v) PEG MME 5000, 0.2 M ammonium sulfate, 0.1 M MES pH 6.7 to yield larger crystals. Crystals with and without inhibitor diffracted to 2.15 and 2.75 angstroms resolution, respectively, and had isomorphous orthorhombic unit cells belonging to space group P2(1)2(1)2(1).

Figure 1

SDS–PAGE of purified BGlu1 fractions from the purification steps for crystallization. Lane 1, Bio-Rad low molecular-weight markers (kDa); lane 2, soluble extract of E. coli cells; lane 3, fusion protein after initial IMAC; lane 4, enterokinase digest; lane 5, BGlu1 after subtractive IMAC; lane 6, BGlu1 after SP-Sepharose, lanes 7 and 8; BGlu1 after the final S200. 5 µg of protein was loaded in all lanes, except lane 7, in which 2.5 µg was loaded.

Figure 2

Crystals of BGlu1 with and without inhibitor crystallized in an orthorhombic crystal form. (a) Plate-shaped tetragonal crystal of BGlu1 with dimensions 120 × 60 × 16 µm grown in 18%(w/v) PEG 8000, 0.2 M zinc acetate dehydrate and 0.1 M sodium cacodylate pH 6.5 by microbatch screening. (b) BGlu1 protein incubated with DNP2FG crystallized as tetragonal crystals, one with dimensions of 320 × 140 × 20 µm, in a hanging-drop vapour-diffusion plate. The precipitant was 23%(w/v) PEG MME 5000, 0.2 M ammonium sulfate, 0.1 M MES pH 6.7.