Open AccessPurification, crystallization and preliminary X‐ray analysis of rice BGlu1 β‐glucosidase with and without 2‐deoxy‐2‐fluoro‐β‐ d ‐glucoside
Author(s) -
Svasti Jisnuson,
Chuenchor Watchalee,
Opassiri Rodjana,
Pengthaisong Salila,
Yuvaniyama Jirundon,
Ketudat Cairns James R.
Publication year - 2006
Publication title -
acta crystallographica section f
Language(s) - English
Resource type - Journals
ISSN - 1744-3091
DOI - 10.1107/s1744309106027084
Subject(s) - crystallization , chemistry , orthorhombic crystal system , affinity chromatography , ammonium sulfate , chromatography , size exclusion chromatography , escherichia coli , crystallography , nuclear chemistry , enzyme , crystal structure , biochemistry , organic chemistry , gene
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 P 2 1 2 1 2 1 .