Synthesis in Escherichia coli of human adenovirus type 12 transforming proteins encoded by early region 1A 13S mRNA and 12S mRNA.
Synthesis in Escherichia coli of human adenovirus type 12 transforming proteins encoded by early region 1A 13S mRNA and 12S mRNA.
复制标题
在大肠杆菌中合成由早期区域 1A 13S mRNA 和 12S mRNA 编码的人腺病毒 12 型转化蛋白。
DOI：
10.1073/pnas.81.20.6300
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发表时间：
1984
影响因子：
11.1
通讯作者：
Green,M
中科院分区：
文献类型：
--
作者：
Kimelman,D;Lucher,LA;Brackmann,KH;Symington,JS;Ptashne,M;Green,M
人腺病毒（Ad）编码的早期区域1A（E1 A）肿瘤（T）抗原与病毒早期基因的正调控、某些细胞基因的正调控和负调控以及细胞永生化和转化有关。为了进一步研究Ad E1 A T抗原并便于其纯化，我们克隆了Ad 12 E1 A 13 S mRNA和12 S mRNA的cDNA拷贝，位于杂合大肠杆菌trp-lac（tac）启动子的下游。在E.通过针对在Ad 12 E1 A DNA序列中编码的合成肽的抗体，将由两种不同的Ad 12 E1 A cDNA构建体中的每一种转化的大肠杆菌细胞免疫沉淀为Mr 47，000蛋白。这两种蛋白均在E.大肠杆菌中的Ad 12 E1 AT抗原是真实的、完整的，因为它们具有（i）从DNA序列预测的Ad 12 E1 A NH 2-末端氨基酸序列，（ii）Ad 12 E1 A COOH-末端序列，如用抗肽抗体免疫沉淀所示;和（iii）分子量和酸性等电点与在Ad 12感染和转化的哺乳动物细胞中合成的E1 A T抗原相似。将T抗原纯化至接近同质，产量为100-200微克/g转化的E. coli细胞。
Human adenovirus (Ad)-encoded early region 1A (E1A) tumor (T) antigens have been implicated in the positive regulation of viral early genes, the positive and negative regulation of some cellular genes, and cell immortalization and transformation. To further study the Ad E1A T antigens and to facilitate their purification, we have cloned cDNA copies of the Ad12 E1A 13S mRNA and 12S mRNA downstream of a hybrid Escherichia coli trp-lac (tac) promoter. Up to 8% of the protein synthesized in E. coli cells transformed by each of the two different Ad12 E1A cDNA constructs were immunoprecipitated as a Mr 47,000 protein by antibody to a synthetic peptide encoded in the Ad12 E1A DNA sequence. Both proteins produced in E. coli appear to be authentic and complete Ad12 E1A T antigens because they possess (i) the Ad12 E1A NH2-terminal amino acid sequence predicted from the DNA sequence; (ii) the Ad12 E1A COOH-terminal sequence, as shown by immunoprecipitation with anti-peptide antibody; and (iii) a molecular weight and an acidic isoelectric point similar to that of the E1A T antigens synthesized in Ad12-infected and transformed mammalian cells. The T antigens were purified to near homogeneity in yields of 100-200 micrograms per g wet weight of transformed E. coli cells.