2-Step purification of the Ku DNA repair protein expressed in Escherichia coli.
The Ku protein is involved in DNA double-strand break repair by non-homologous end-joining (NHEJ), which is crucial to the maintenance of genomic integrity in mammals. To study the role of Ku in NHEJ we developed a bicistronic Escherichia coli expression system for the Ku70 and Ku80 subunits. Association of the Ku70 and Ku80 subunits buries a substantial amount of surface area (approximately 9000 A2 [J.R. Walker, R.A. Corpina, J. Goldberg, Structure of the Ku heterodimer bound to DNA and its implications for double-strand break repair, Nature 412 (2001) 607-614]), which suggests that herterodimerization may be important for protein stability. N-terminally His6-tagged Ku80 was soluble in the presence, but not in the absence, of bicistronically expressed untagged Ku70. In a 2-step purification, metal chelating affinity chromatography was followed by step-gradient elution from heparin-agarose. Co-purification of equimolar amounts of His6-tagged Ku80 and untagged Ku70 was observed, which indicated heterodimerization. Recombinant Ku bound dsDNA, activated the catalytic subunit of the DNA-dependent kinase (DNA-PKcs) and functioned in NHEJ reactions in vitro. Our results demonstrate that while the heterodimeric interface of Ku is extensive it is nonetheless possible to produce biologically active Ku protein in E. coli.
Hanakahi
Les A
LA
Department of Biochemistry and Molecular Biology, Johns Hopkins University, Bloomberg School of Public Health, 615 North Wolfe Street, Baltimore, MD 21205, USA. lhanakah@jhsph.edu
D003001:Cloning, Molecular; D004265:DNA Helicases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D004260:DNA Repair; D019281:Dimerization; D004926:Escherichia coli / Q000235:genetics*; D006801:Humans; D000072200:Ku Autoantigen; D008958:Models, Molecular; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D021122:Protein Subunits / Q000235:genetics / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D012995:Solubility
D021122:Protein Subunits; D011994:Recombinant Proteins; D004265:DNA Helicases; C106893:XRCC5 protein, human; D000072200:Ku Autoantigen