Immunoprecipitation of some forms of simian virus 40 large-T antigen by antibodies to synthetic peptides.
Antibodies were raised against six synthetic peptides corresponding to overlapping amino acid sequences (106 through 145) from a putative DNA binding domain in simian virus 40 (SV40) large-T antigens. All six antipeptide sera immunoprecipitated large-T from crude extracts of SV40-transformed cells, but the efficiency varied widely; in general, antibodies to the longer peptides produced the strongest anti-large-T activity. Antisera were purified by immunoaffinity chromatography on immobilized peptide. The purified antisera recognized only some forms of large-T; full-sized large-T from transformed cells, super-T from SV3T3 C120 cells, and 70,000-dalton T-antigen from Taq-BamHI cells were immunoprecipitated, whereas large-T from productively infected cells reacted irreproducibly, and the full-sized protein, synthesized in vitro or eluted from sodium dodecyl sulfate-containing gels, and the 33,000- and 22,000-dalton truncated large-Ts from Swiss SV3T3 and MES2006 cells, respectively, were not immunoprecipitated. This pattern of reactivity was explained when extracts were fractionated by sucrose density centrifugation, and it was found that only rapidly sedimenting forms of large-T were immunoprecipitated by the antipeptide sera; that is, large-T complexed with nonviral T antigen was detected, whereas lighter forms were not detected. Cascade immunoprecipitations did not support the view that this result was caused by the low affinity of the peptide antisera for large-T, and Western blotting experiments confirmed that the peptide antisera react directly with immobilized, monomeric large-T but not with nonviral T antigen. Immunoprecipitation assays to detect large-T:nonviral T antigen complexes bound specifically to fragments of SV40 DNA showed that under conditions of apparent antibody excess, DNA still bound to the complex.
D000818:Animals; D000906:Antibodies / Q000302:isolation & purification; D000936:Antigen-Antibody Complex / Q000032:analysis*; D000952:Antigens, Polyomavirus Transforming; D001665:Binding Sites; D002460:Cell Line; D051379:Mice; D010455:Peptides / Q000276:immunology; D010957:Plasmids; D011485:Protein Binding; D013539:Simian virus 40 / Q000235:genetics*; D013724:Teratoma; D014764:Viral Proteins / Q000032:analysis* / Q000276:immunology
D000906:Antibodies; D000936:Antigen-Antibody Complex; D000952:Antigens, Polyomavirus Transforming; D010455:Peptides; D014764:Viral Proteins