Purification and molecular characterization of adenovirus type 2 DNA-binding protein
- 31 December 1976
- journal article
- research article
- Published by American Society for Microbiology in Journal of Virology
- Vol. 21 (1), 338-346
- https://doi.org/10.1128/jvi.21.1.338-346.1977
Abstract
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 .mu.g of binding protein (95-99% homogeneity) starting with 2 .times. 109 infected [human oral carcinoma KB] cells. By omitting the Sephadex G-200 step, 400-600 .mu.g 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 retain its native state as indicated by binding to single-stranded but not native Ad2 DNA, almost complete precipitation by immunoglobulin G from hamsters immunized by extracts of tumors [hamster] induced by Ad2 SV-40 hybrid viruses, and 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 2 values, a MW 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.This publication has 21 references indexed in Scilit:
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