Open AccessExpression, purification and preliminary crystallographic analysis of 2,4‐dihydroxy‐hepta‐2‐ene‐1,7‐dioate aldolase (HpcH) from Escherichia coli C
Author(s) -
Rea Dean,
Bugg Timothy D. H.,
Fülöp Vilmos,
Roper David I.
Publication year - 2005
Publication title -
acta crystallographica section f
Language(s) - English
Resource type - Journals
ISSN - 1744-3091
DOI - 10.1107/s1744309105023079
Subject(s) - escherichia coli , crystallization , aldolase a , sodium , crystallography , chemistry , tris , enzyme , nuclear chemistry , biochemistry , gene , organic chemistry
The gene encoding 2,4‐dihydroxy‐hepta‐2‐ene‐1,7‐dioate (HHED) aldolase (HpcH; EC 4.1.2) from Escherichia coli C was cloned into the high‐expression plasmid pProEx‐HTa and overexpressed in E. coli BL21 (DE3). The 28 kDa enzyme was purified using immobilized metal‐affinity and size‐exclusion chromatography prior to crystallization. Crystals were obtained by the hanging‐drop vapour‐diffusion method at 277 K from a number of screening conditions. Type I crystals grown in a solution containing 0.4 M ammonium dihydrogen phosphate belong to space group R 32, with unit‐cell parameters a = b = 128.92, c = 175.30 Å. Type II crystals grown in a solution containing 0.5 M sodium chloride, 0.1 M sodium citrate pH 5.5 belong to space group I 222, with unit‐cell parameters a = 133.39, b = 155.39, c = 168.80 Å. Complete data sets were collected to 1.6 and 2.0 Å from type I and type II crystals, respectively, using synchrotron radiation.