Purification, crystallization and preliminary X-ray analysis of rice BGlu1 beta-glucosidase with and without 2-deoxy-2-fluoro-beta-D-glucoside.
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).
Chuenchor
Watchalee
W
School of Chemistry and Biochemistry, Institute of Science, Suranaree University of Technology, University Avenue, Muang District, Nakhon Ratchasima 30000, Thailand.
Pengthaisong
Salila
S
Yuvaniyama
Jirundon
J
Opassiri
Rodjana
R
Svasti
Jisnuson
J
Ketudat Cairns
James R
JR
D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D003460:Crystallization; D004926:Escherichia coli / Q000201:enzymology; D005960:Glucosides / Q000737:chemistry* / Q000378:metabolism; D012275:Oryza / Q000201:enzymology*; D010940:Plant Proteins / Q000737:chemistry / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D014961:X-Ray Diffraction; D001617:beta-Glucosidase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism
D005960:Glucosides; D010940:Plant Proteins; D011994:Recombinant Proteins; D001617:beta-Glucosidase