Studies on epstein‐barr virus‐related antigens. III. Purification of the virus‐determined nuclear antigen (EBNA) from non‐producer raji cells

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Abstract
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° 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.