title: Expression, purification, crystallization and preliminary characterization of an HHED aldolase homologue from Escherichia coli K12.
abstract: An ORF designated b2245 (yfaU) in the Escherichia coli K12 genome sequence, identified as an HHED aldolase homologue, was cloned into the high-expression plasmid pT7-7 and overexpressed in E. coli B835(DE3). The enzyme was purified in three steps to 95% purity prior to crystallization. Crystals were obtained by the hanging-drop vapour-diffusion method at 277 K from a number of screening conditions. Crystals suitable for structural studies were grown from solutions containing 0.4 M ammonium dihydrogen phosphate and grew to a maximum dimension of approximately 0.5 mm. Diffraction data to 1.7 A were collected using an in-house Cu Kalpha radiation source at 100 K. The crystals belong to space group C222(1), with unit-cell parameters a = 105.1, b = 136.6, c = 123.1 A. A 90% complete data set was collected to 1.78 A from a single native crystal using in-house facilities.
authors.affiliation: Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, England.
authors.forename: Anne
authors.initials: A
authors.lastname: Wright
authors.forename: Anne
authors.initials: A
authors.lastname: Blewett
authors.forename: Vilmos
authors.initials: V
authors.lastname: Fulop
authors.forename: Ronald
authors.initials: R
authors.lastname: Cooper
authors.forename: Shirley
authors.initials: S
authors.lastname: Burrows
authors.forename: Christopher
authors.initials: C
authors.lastname: Jones
authors.forename: David
authors.initials: D
authors.lastname: Roper
mesh_terms: D000446:Aldehyde-Lyases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D003001:Cloning, Molecular; D003460:Crystallization; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D011487:Protein Conformation
chemical_list: D029968:Escherichia coli Proteins; C097246:2,4-dihydroxyhept-2-ene-1,7-dioic acid aldolase, E coli; D000446:Aldehyde-Lyases