Kimelman D, Lucher L A, Brackmann K H, Symington J S, Ptashne M, Green M
Proc Natl Acad Sci U S A. 1984 Oct;81(20):6300-4. doi: 10.1073/pnas.81.20.6300.
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.
人腺病毒（Ad）编码的早期区域1A（E1A）肿瘤（T）抗原与病毒早期基因的正调控、某些细胞基因的正负调控以及细胞永生化和转化有关。为了进一步研究Ad E1A T抗原并便于其纯化，我们已将Ad12 E1A 13S mRNA和12S mRNA的cDNA拷贝克隆到杂交大肠杆菌色氨酸-乳糖（tac）启动子的下游。在由两种不同的Ad12 E1A cDNA构建体之一转化的大肠杆菌细胞中合成的蛋白质中，高达8%被Ad12 E1A DNA序列中编码的合成肽抗体免疫沉淀为分子量47,000的蛋白质。在大肠杆菌中产生的这两种蛋白质似乎都是真实且完整的Ad12 E1A T抗原，因为它们具有：（i）根据DNA序列预测的Ad12 E1A NH2末端氨基酸序列；（ii）抗肽抗体免疫沉淀显示的Ad12 E1A COOH末端序列；以及（iii）与在Ad12感染和转化的哺乳动物细胞中合成的E1A T抗原相似的分子量和酸性等电点。这些T抗原被纯化至接近均一，每克湿重转化的大肠杆菌细胞产量为100 - 200微克。
