Open AccessExpression, purification, crystallization and preliminary X‐ray crystallographic studies of Deinococcus radiodurans thioredoxin reductase
Author(s) -
Obiero Josiah,
Bonderoff Sara A.,
Goertzen Meghan M.,
Sanders David A. R.
Publication year - 2006
Publication title -
acta crystallographica section f
Language(s) - English
Resource type - Journals
ISSN - 1744-3091
DOI - 10.1107/s1744309106024845
Subject(s) - deinococcus radiodurans , escherichia coli , thioredoxin , thioredoxin reductase , fumarate reductase , oxidoreductase , molecular replacement , crystallography , chemistry , biochemistry , reductase , geobacillus stearothermophilus , enzyme , dna , thermophile , gene
Deinococcus radiodurans , a Gram‐positive bacterium capable of withstanding extreme ionizing radiation, contains two thioredoxins (Trx and Trx1) and a single thioredoxin reductase (TrxR) as part of its response to oxidative stress. Thioredoxin reductase is a member of the family of pyridine nucleotide‐disulfide oxidoreductase flavoenzymes. Recombinant D. radiodurans TrxR with a His tag at the N‐terminus was expressed in Escherichia coli and purified by metal‐affinity chromatography. The protein was crystallized using the sitting‐drop vapour‐diffusion method in the presence of 35% PEG 4000, 0.2 M ammonium acetate and citric acid buffer pH 5.1 at 293 K. X‐ray diffraction data were collected on a cryocooled crystal to a resolution of 1.9 Å using a synchrotron‐radiation source. The space group was determined to be P 3 2 21, with unit‐cell parameters a = b = 84.33, c = 159.88 Å. The structure of the enzyme has been solved by molecular‐replacement methods and structure refinement is in progress.