Connors R W, Sweet R W, Noveral J P, Pfarr D S, Trill J J, Shebuski R J, Berkowitz B A, Williams D, Franklin S, Reff M E
Department of Molecular Genetics, Smith Kline and French Laboratories, King of Prussia, PA 19406-0939.
DNA. 1988 Nov;7(9):651-61. doi: 10.1089/dna.1988.7.651.
High-level expression of human tissue-type plasminogen activator was accomplished in endothelial cells by a novel approach to dihydrofolate reductase (DHFR) coamplification in DHFR+ cells. A tripartite mammalian expression vector coding for DHFR, neomycin phosphotransferase, and the t-PA gene was introduced into bovine endothelial cells by transfection and selection for G418 resistance. Upon methotrexate selection of these transformants, we obtained endothelial cells that had amplified the plasmid-encoded DHFR and t-PA genes. As a result, cell lines were isolated that efficiently produced t-PA (greater than 4 pg/cell.day). This t-PA was purified and compared with recombinant t-PA produced in Chinese hamster ovary cells. These two t-PA samples differed in carbohydrate composition, and amounts of 530 and 527 amino acid forms but had similar in vitro activity.
通过一种在二氢叶酸还原酶（DHFR）阳性细胞中进行二氢叶酸还原酶共扩增的新方法，在人内皮细胞中实现了人组织型纤溶酶原激活剂的高水平表达。通过转染并选择对G418有抗性，将编码二氢叶酸还原酶、新霉素磷酸转移酶和t-PA基因的三方哺乳动物表达载体导入牛内皮细胞。在对这些转化体进行甲氨蝶呤选择后，我们获得了扩增了质粒编码的二氢叶酸还原酶和t-PA基因的内皮细胞。结果，分离出了能高效产生t-PA（大于4 pg/细胞·天）的细胞系。这种t-PA被纯化，并与在中国仓鼠卵巢细胞中产生的重组t-PA进行比较。这两种t-PA样品在碳水化合物组成以及530和527个氨基酸形式的含量上有所不同，但体外活性相似。
