Purification, crystallization and preliminary X-ray analysis of rice BGlu1 β-glucosidase with and without 2-deoxy-2-fluoro-β-d-glucoside
- School of Chemistry and Biochemistry, Institute of Science, Suranaree University of Technology, University Avenue, Muang District, Nakhon Ratchasima 30000 (Thailand)
- Department of Biochemistry and Center for Excellence in Protein Structure and Function, Faculty of Science, Mahidol University, Rama 6 Road, Phayathai, Bangkok 10400 (Thailand)
Rice BGlu1 β-glucosidase was purified from recombinant E. coli and crystallized with and without the inhibitor 2-deoxy-2-fluoro-β-d-glucose. The crystals diffracted to 2.15 and 2.75 Å, respectively. 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 P2{sub 1}2{sub 1}2{sub 1}.
- OSTI ID:
- 22360167
- Journal Information:
- Acta Crystallographica. Section F, Vol. 62, Issue Pt 8; Other Information: PMCID: PMC2242908; PMID: 16880561; PUBLISHER-ID: bo5003; OAI: oai:pubmedcentral.nih.gov:2242908; Copyright (c) International Union of Crystallography 2006; Country of input: International Atomic Energy Agency (IAEA); ISSN 1744-3091
- Country of Publication:
- United Kingdom
- Language:
- English
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