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首页> 外文期刊>Journal of Virology >Purification and molecular characterization of adenovirus type 2 DNA-binding protein.
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Purification and molecular characterization of adenovirus type 2 DNA-binding protein.

机译：腺病毒型2 DNA结合蛋白的纯化和分子表征。

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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.
机译：通过顺序DNA-纤维素，Sephadex G-200和DEAE-Sephadex色谱法纯化腺病毒型2（AD2）DNA结合蛋白，从2 x开始，120杯结合蛋白（95-99％均匀性）的产率为120米。 10（9）感染细胞。通过省略Sephadex G-200步骤，获得400至600杯的95％纯结合蛋白。为了获得高产高产高产高度纯化的结合蛋白质，在制备期间，必须在选定的阶段包括在选定的阶段中的脱氧胆酸盐和非甲酸p-40。高纯化的结合蛋白似乎保留了其天然阶段，如下所示：（i）与单链但不是天然Ad2 DNA的结合，（II）通过AD2-诱导的肿瘤提取物免疫的免疫球蛋白G几乎完全沉淀Simian病毒40杂交病毒，（III）仅具有从DNA-纤维素色谱法获得的结合蛋白质的相同沉降系数。在蔗糖梯度和凝胶过滤中的区间离心显示，纯化的结合蛋白具有3.4s的沉降系数和5.2nm的斯托克斯半径。基于这两个值，计算了73,000的分子量，同时从十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳估计。计算摩擦比为1.88，表明AD2 DNA结合蛋白没有典型的球状蛋白质结构。

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