Studies on Epstein-Barr virus-related antigens. III. Purification of the virus-determined nuclear antigen (EBNA) from non-producer Raji cells.
The purification of Epstein-Barr virus-determined nuclear antigen (EBNA) was attempted on the basis of its biochemical and physicochemical properties and its immunological specificity, assayed by the indirect single radial immunodiffusion test and anti-complement immunofluorescence absorption test. When non-producer Raji cell extract was subjected to 20--60% saturated ammonium sulfate fractionation, followed by DNA-cellulose chromatography and immunoadsorbent chromatography, EBNA was purified more than 3,000 times with a 8% yield. Such purified protein was composed of three polypeptides with molecular weights of 100,000 +/- 5,000, 70,000 +/- 5,000 and 50,000 daltons, respectively, on SDS-polyacrylamide gel electrophoresis. Similar purification was achieved by heating the extract at 70 degrees C for 10 min, instead of ammonium sulfate fractionation, followed by DNA-cellulose chromatography and immunoadsorbent chromatography. This final preparation consisted almost exclusively of 100,000 +/- 5,000 daltons polypeptide, 50,000 polypeptide, and the 70,000 +/- 5,000 polypeptide passing through the adsorbent column. These findings suggest that EBNA is probably a molecular complex of three smaller subunits of heat-stable 100,000 +/- 5,000 and 50,000 daltons and heat-labile 70,000 +/- 5,000 daltons polypeptides, respectively.
D000645:Ammonium Sulfate; D000956:Antigens, Viral / Q000302:isolation & purification*; D002458:Cell Fractionation; D002845:Chromatography; D003168:Complement Fixation Tests; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes; D005455:Fluorescent Antibody Technique; D004854:Herpesvirus 4, Human / Q000276:immunology*; D006358:Hot Temperature; D006801:Humans; D007106:Immune Sera; D005779:Immunodiffusion; D008970:Molecular Weight; D014764:Viral Proteins / Q000032:analysis
D000956:Antigens, Viral; D000939:Epitopes; D007106:Immune Sera; D014764:Viral Proteins; D000645:Ammonium Sulfate