Expression, purification, crystallization and preliminary characterization of an HHED aldolase homologue from Escherichia coli K12.
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.
Wright
Anne
A
Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, England.
Blewett
Anne
A
Fulop
Vilmos
V
Cooper
Ronald
R
Burrows
Shirley
S
Jones
Christopher
C
Roper
David
D
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
D029968:Escherichia coli Proteins; C097246:2,4-dihydroxyhept-2-ene-1,7-dioic acid aldolase, E coli; D000446:Aldehyde-Lyases