Purification of a native membrane-associated adenovirus tumor antigen.
A 15,000-dalton protein was purified from HeLa cells infected with adenovirus type 2. Proteins solubilized from a membrane fraction of lytically infected cells was used as the starting material for purification. Subsequent purification steps involved lentil-lectin, phosphocellulose, hydroxyapatite, DEAE-cellulose, and aminohexyl-Sepharose chromatographies. A monospecific antiserum, raised against the purified protein, immunoprecipitated a 15,000-dalton protein encoded in early-region E1B (E1B/15K protein) of the adenovirus type 2 DNA. Tryptic finger print analysis revealed that the purified protein was identical to the E1B/15K protein encoded in the transforming part of the viral genome. The antiserum immunoprecipitated the E1B/15K protein from a variety of viral transformed cell lines isolated from humans, rats, or hamsters. The E1B/15K protein was associated with the membrane fraction of both lytically and virus-transformed cell lines and could only be released by detergent treatment. Furthermore, a 11,000- to 12,000-dalton protein that could be precipitated with the anti-E1B/15K serum was recovered from membranes treated with trypsin or proteinase K, suggesting that a major part of the E1B/15K protein is protected in membrane vesicles. Translation of early viral mRNA in a cell-free system, supplemented with rough microsomes, showed that this protein was associated with the membrane fraction also in vitro.
D000260:Adenoviruses, Human / Q000235:genetics / Q000276:immunology*; D000956:Antigens, Viral / Q000235:genetics / Q000302:isolation & purification*; D000957:Antigens, Viral, Tumor; D002472:Cell Transformation, Viral; D002845:Chromatography; D005814:Genes, Viral; D006367:HeLa Cells; D006801:Humans; D008565:Membrane Proteins / Q000302:isolation & purification; D008566:Membranes / Q000276:immunology*; D008970:Molecular Weight; D014764:Viral Proteins / Q000302:isolation & purification
D000956:Antigens, Viral; D000957:Antigens, Viral, Tumor; D008565:Membrane Proteins; D014764:Viral Proteins