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

• Published in: Acta crystallographica. Section F, Structural biology and crystallization communications Vol. 62; no. 8; pp. 798 - 801
• Main Authors: Svasti, Jisnuson, Chuenchor, Watchalee, Opassiri, Rodjana, Pengthaisong, Salila, Yuvaniyama, Jirundon, Ketudat Cairns, James R.
• Format: Journal Article
• Language: English
• Published: 5 Abbey Square, Chester, Cheshire CH1 2HU, England Blackwell Publishing Ltd 01.08.2006; International Union of Crystallography
• Subjects: 2,4‐dinitrophenyl‐2‐­deoxy‐2‐fluoro‐β‐d‐glucopyranoside; 4-dinitrophenyl-2-­deoxy-2-fluoro-β-d-glucopyranoside; ACETATES; AFFINITY; AMMONIUM SULFATES; beta-Glucosidase - antagonists & inhibitors; beta-Glucosidase - chemistry; beta-Glucosidase - isolation & purification; beta-Glucosidase - metabolism; BGlu1 β-glucosidase; Chromatography, Affinity; Cloning, Molecular; CONDENSED MATTER PHYSICS, SUPERCONDUCTIVITY AND SUPERFLUIDITY; CRYSTALLIZATION; Crystallization Communications; CRYSTALS; DIFFUSION; ESCHERICHIA COLI; Escherichia coli - enzymology; GELS; GLUCOSE; Glucosides - chemistry; Glucosides - metabolism; IMPURITIES; Oryza - enzymology; Oryza sativa; Plant Proteins - chemistry; Plant Proteins - isolation & purification; Recombinant Proteins - chemistry; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; RESOLUTION; rice; SODIUM; SPACE GROUPS; X-Ray Diffraction; ZINC
• ISSN: 1744-3091; 1744-3091
• DOI: 10.1107/S1744309106027084

Rice (Oryza sativa) BGlu1 β‐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‐β‐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 Å resolution, respectively, and had isomorphous orthorhombic unit cells belonging to space group P212121.