Expression and isotopic labeling of structural domains of the human protein DEK.
The 375 amino acid human protein DEK has been expressed in two functional, structured domains. DEK is an abundant nuclear protein that associates with chromatin and alters its topology by introducing positive supercoiling in DNA, which results in lower replication efficiency. DEK has clinical importance as transfection of the cDNA of the C-terminal region of DEK can partially reverse the abnormal DNA-mutagen sensitivity in fibroblasts derived from ataxia-telangiectasia (A-T) patients, and elevated levels of DEK mRNA are observed in various forms of cancer. Because high-level expression of full-length DEK has proved elusive, we sought an alternative for structural studies that would provide insights on DEK's function. We have discovered that DEK contains two structured domains and have expressed these domains at a high level in Escherichia coli in M9 minimal media. The N-terminal domain (amino acids 68-226) includes the region responsible for introducing supercoils into DNA, and the C-terminal domain (amino acids 309-375) includes the region that can reverse the abnormal DNA-mutagen sensitivity of A-T cells. 1H-15N correlation nuclear magnetic resonance spectra of these two fragments reveal the characteristic signature of folded proteins.
Devany
Matthew
M
Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN 55455, USA.
Kotharu
N Prasad
NP
Matsuo
Hiroshi
H
D002868:Chromosomal Proteins, Non-Histone / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D003001:Cloning, Molecular / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D007553:Isotope Labeling; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular; D015513:Oncogene Proteins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics; D000075223:Poly-ADP-Ribose Binding Proteins; D017434:Protein Structure, Tertiary
D002868:Chromosomal Proteins, Non-Histone; C084738:DEK protein, human; D009587:Nitrogen Isotopes; D015513:Oncogene Proteins; D010446:Peptide Fragments; D000075223:Poly-ADP-Ribose Binding Proteins