Open AccessExpression, purification, crystallization and preliminary X‐ray analysis of rice ( Oryza sativa L.) Os4BGlu12 β‐glucosidase
Author(s) -
Sansenya Sompong,
Ketudat Cairns James R.,
Opassiri Rodjana
Publication year - 2010
Publication title -
acta crystallographica section f
Language(s) - English
Resource type - Journals
ISSN - 1744-3091
DOI - 10.1107/s174430911000103x
Subject(s) - oryza sativa , crystallization , polyethylene glycol , escherichia coli , chemistry , hydrolase , protein data bank (rcsb pdb) , crystallography , thioredoxin , molecular replacement , tris , crystal structure , stereochemistry , enzyme , biochemistry , organic chemistry , gene
Rice ( Oryza sativa L.) Os4BGlu12, a glycoside hydrolase family 1 β‐glucosidase (EC 3.2.1.21), was expressed as a fusion protein with an N‐terminal thioredoxin/His 6 tag in Escherichia coli strain Origami B (DE3) and purified with subsequent removal of the N‐terminal tag. Native Os4BGlu12 and its complex with 2,4‐dinitrophenyl‐2‐deoxy‐2‐fluoro‐β‐ d ‐glucopyranoside (DNP2FG) were crystallized using 19% polyethylene glycol (3350 or 2000, respectively) in 0.1 M Tris–HCl pH 8.5, 0.16 M NaCl at 288 K. Diffraction data sets for the apo and inhibitor‐bound forms were collected to 2.50 and 2.45 Å resolution, respectively. The space group and the unit‐cell parameters of the crystal indicated the presence of two molecules per asymmetric unit, with a solvent content of 50%. The structure of Os4BGlu12 was successfully solved in space group P 4 3 2 1 2 by molecular replacement using the white clover cyanogenic β‐glucosidase structure (PDB code 1cbg ) as a search model.