Open AccessExpression, purification and crystallization of 2‐oxo‐hept‐4‐ene‐1,7‐dioate hydratase (HpcG) from Escherichia coli C
Author(s) -
Rea Dean,
Adachi Tomoko,
Izumi Atsushi,
Park SamYong,
Tame Jeremy R. H.,
Roper David I.
Publication year - 2006
Publication title -
acta crystallographica section f
Language(s) - English
Resource type - Journals
ISSN - 1744-3091
DOI - 10.1107/s1744309106035901
Subject(s) - escherichia coli , crystallization , chemistry , biochemistry , gene , organic chemistry
The gene encoding 2‐oxo‐hept‐3‐ene‐1,7‐dioic acid (OHED) hydratase (HpcG) was cloned into the high‐expression plasmid pET26b and overexpressed in Escherichia coli BL21(DE3). The enzyme was purified in three steps to greater than 95% purity prior to crystallization. Crystals were obtained by the hanging‐drop vapour‐diffusion method at 277 K in a number of screening conditions. Crystals measuring up to 1.5 mm in their longest dimension were grown from solutions containing polyethylene glycol 20 000. The crystals belonged to space group P 4 1 2 1 2 or P 4 3 2 1 2, with unit‐cell parameters a = 136, b = 136, c = 192 Å. A complete data set was collected to 2.1 Å from a single cryocooled crystal at 100 K using synchrotron radiation.