Production and characterization of recombinant human Ku antigen.
Ku is an ubiquitous nuclear heterodimeric protein consisting of p70 and p86 subunits that binds double-stranded DNA termini and associates with chromosomes in vivo. It was originally described as an autoantigen in patients with certain autoimmune diseases. The individual subunits of Ku have been difficult to isolate from human cells without denaturation and attempts to produce functional recombinant Ku have been largely unsuccessful. Here, we utilize two recombinant baculoviral vectors that carry p70 or p86 cDNA and express the Ku subunits individually as well as assemble them into the complete Ku heterodimer. In an electrophoretic mobility shift assay, recombinant Ku binds to linear double-stranded DNA but not to supercoiled, nicked circular, nor linear single-stranded DNA. Neither subunit binds DNA by itself indicating that heterodimerization is essential for function. We also describe a simple purification method for the isolation of highly purified recombinant Ku using a hexahistidine tag. The baculovirus expression system provides a stable and efficient source of not only the p70 and p86 subunits but also the functional Ku heterodimer.
Ono
M
M
Department of Microbiology, University of Texas Southwestern Medical Center, Dallas 75235.
Tucker
P W
PW
Capra
J D
JD
D000818:Animals; D034961:Antigens, Nuclear; D001324:Autoantigens / Q000737:chemistry / Q000235:genetics*; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D002460:Cell Line; D004247:DNA / Q000378:metabolism; D004265:DNA Helicases; D004270:DNA, Circular / Q000378:metabolism; D018076:DNA, Complementary / Q000235:genetics; D004277:DNA, Single-Stranded / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D000072200:Ku Autoantigen; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis; D018411:Spodoptera / Q000378:metabolism; D014162:Transfection
D034961:Antigens, Nuclear; D001324:Autoantigens; D004270:DNA, Circular; D018076:DNA, Complementary; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; D009687:Nuclear Proteins; D011994:Recombinant Proteins; D004247:DNA; D004265:DNA Helicases; C106893:XRCC5 protein, human; C000606509:Xrcc6 protein, human; D000072200:Ku Autoantigen