Purification and molecular characterization of adenovirus type 2 DNA-binding protein.
An adenovirus type 2 (Ad2) DNA-binding protein was purified by sequential DNA-cellulose, Sephadex G-200, and DEAE-Sephadex chromatography, with a yield of 120 mug of binding protein (95 to 99% homogeneity) starting with 2 X 10(9) infected cells. By omitting the Sephadex G-200 step, 400 to 600 mug of 95% pure binding protein was obtained. To obtain high yields of highly purified binding protein, it was necessary to include deoxycholate and Nonidet P-40 at selected stages during the preparation. The highly purified binding protein appeared to have retained its native stage as indicated by: (i) binding to single-stranded but not native Ad2 DNA, (ii) almost complete precipitation by immunoglobulin G from hamsters immunized by extracts of tumors induced by Ad2-simian virus 40 hybrid viruses, and (iii) identical sedimentation coefficient with binding protein obtained from DNA-cellulose chromatography only. Zonal centrifugation in sucrose gradients and gel filtration revealed that purified binding protein has a sedimentation coefficient of 3.4S and a Stokes radius of 5.2 nm. Based on these two values, a molecular weight of 73,000 was calculated, in agreement with the estimate from sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A frictional ratio of 1.88 was calculated, suggesting that the Ad2 DNA-binding protein does not have a typical globular protein structure.
D000260:Adenoviruses, Human / Q000032:analysis* / Q000276:immunology / Q000378:metabolism; D002845:Chromatography; D004277:DNA, Single-Stranded / Q000378:metabolism; D004279:DNA, Viral / Q000378:metabolism; D000939:Epitopes; D008970:Molecular Weight; D011485:Protein Binding; D014764:Viral Proteins / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism
D004277:DNA, Single-Stranded; D004279:DNA, Viral; D000939:Epitopes; D014764:Viral Proteins