Expression, purification and crystallization of 2-oxo-hept-4-ene-1,7-dioate hydratase (HpcG) from Escherichia coli C

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 P4(1)2(1)2 or P4(3)2(1)2, with unit-cell parameters a = 136, b = 136, c = 192 A. A complete data set was collected to 2.1 A from a single cryocooled crystal at 100 K using synchrotron radiation.