Identification and purification of a protein encoded by the human adenovirus type 2 transforming region.
Identification and purification of a protein encoded by the human adenovirus type 2 transforming region.
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人腺病毒2型转化区编码的蛋白质的鉴定和纯化。
DOI：
10.1128/jvi.42.1.30-41.1982
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发表时间：
1982
影响因子：
5.4
通讯作者：
Matsuo,T
中科院分区：
文献类型：
--
作者：
Green,M;Brackmann,KH;Cartas,MA;Matsuo,T
人腺病毒2型（Ad2）转化基因位于早期区域E1a（图位1.3 ~ 4.5）和E1b（图位4.6 ~ 11.2）。我们已经鉴定并纯化了一个主要的20,000分子量（20K）蛋白，并表明它是由E1b编码的。使用含有e1b编码蛋白抗体的Ad2转化细胞抗血清，我们从核质中免疫沉淀53K和19K蛋白，并从Ad2有效感染和Ad2转化细胞的细胞质S-100部分中免疫沉淀53K、19K和20K蛋白。19K蛋白存在于核质和细胞质中，而20K蛋白只存在于细胞质中。53K和19K蛋白是已知的ad2e1b编码蛋白。通过deae - sepacel层析和反相高效液相层析纯化20K蛋白，纯度接近均匀，产率为20% ~ 50%。纯化后的20K蛋白通过反相高效液相色谱法发现，其蛋氨酸标记的色氨酸肽大部分与E1b-53K相同，因此与53K蛋白密切相关。19K蛋白似乎不与20K或53K蛋白共享色氨酸。为了提供更直接的证据证明20K蛋白是病毒编码的，我们在体外翻译了e1b特异性mRNA。翻译产物的免疫沉淀分析和高效液相色谱纯化鉴定出20K蛋白与从感染细胞和转化细胞中分离的20K蛋白具有相同的胰蛋白酶肽。这些发现表明ad220k蛋白是ad2e1b mRNA的主要翻译产物。
The human adenovirus type 2 (Ad2) transforming genes are located in early regions E1a (map position 1.3 to 4.5) and E1b (map position 4.6 to 11.2). We have identified and purified to near homogeneity a major 20,000-molecular-weight (20K) protein and have shown that it is coded by E1b. Using an Ad2-transformed cell antiserum which contained antibody to E1b-coded proteins, we immunoprecipitated 53K and 19K proteins from the nucleoplasm and 53K, 19K, and 20K proteins from the cytoplasmic S-100 fraction of Ad2 productively infected and Ad2-transformed cells. The 19K protein was present in both the nucleoplasm and the cytoplasm, whereas the 20K protein was found only in the cytoplasm. The 53K and 19K proteins are known Ad2 E1b-coded proteins. The 20K protein was purified to near homogeneity in 20 to 50% yields by sequential DEAE-Sephacel chromatography and reverse-phase high-performance liquid chromatography. Purified 20K protein shares most of its methionine-labeled tryptic peptides with E1b-53K, as shown by reverse-phase high-performance liquid chromatography, and therefore is closely related to the 53K protein. The 19K protein does not appear to share tryptic peptides with either 20K or 53K protein. To provide more direct evidence that 20K protein is virus-coded, we translated E1b-specific mRNA in vitro. Both immunoprecipitation analysis and high-performance liquid chromatography purification of the translated product identified a 20K protein that has the same tryptic peptides as the 20K protein isolated from infected and from transformed cells. These findings suggest that the Ad2 20K protein is a primary translation product of an Ad2 E1b mRNA.