Characterization of the 70KDA component of the human Ku autoantigen expressed in insect cell nuclei using a recombinant baculovirus vector.
The Ku autoantigen is a human nuclear, DNA-binding heterodimer of 70kDa and 86kDa proteins. It is the target of autoantibodies in several autoimmune diseases. We now report the expression of a cDNA encoding the 70kDa Ku protein. Large amounts of protein were obtained using a recombinant baculovirus vector, in contrast with earlier unsuccessful attempts using other expression systems. We demonstrate that the 70kDa Ku protein is targeted to the nucleus and is associated with the nuclear matrix when expressed in the absence of the 86kDa Ku component. No post-translational modifications were observed. The 70kDa protein binds double and single-stranded DNA with very high affinity. Our results suggest that the baculovirus expression system may be of widespread use in the production and characterization of human autoantigens.
Allaway
G P
GP
Laboratory of Oral Medicine, National Institute of Dental Research, National Institutes of Health, Bethesda, MD 20892.
Vivino
A A
AA
Kohn
L D
LD
Notkins
A L
AL
Prabhakar
B S
BS
D000818:Animals; D034961:Antigens, Nuclear; D000954:Antigens, Surface / Q000235:genetics*; D001324:Autoantigens / Q000235:genetics*; D002467:Cell Nucleus / Q000378:metabolism*; D004247:DNA / Q000032:analysis; D004265:DNA Helicases; D004268:DNA-Binding Proteins / Q000235:genetics*; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D066298:In Vitro Techniques; D000072200:Ku Autoantigen; D007915:Lepidoptera / Q000235:genetics* / Q000276:immunology; D008970:Molecular Weight; D011499:Protein Processing, Post-Translational; D012313:RNA / Q000032:analysis; D011995:Recombination, Genetic; D014162:Transfection
D034961:Antigens, Nuclear; D000954:Antigens, Surface; D001324:Autoantigens; D004268:DNA-Binding Proteins; D012313:RNA; D004247:DNA; D004265:DNA Helicases; C106893:XRCC5 protein, human; C000606509:Xrcc6 protein, human; D000072200:Ku Autoantigen