Langer G, Toschi L, Dieckmann J, Schleuning W D
Research Laboratories of Schering AG Berlin, Germany.
Gene. 1995 Aug 19;161(2):287-92. doi: 10.1016/0378-1119(95)00220-z.
A reporter plasmid coding for the low-molecular-weight (low-M(r)) form of single-chain urokinase-type plasminogen activator (low-M(r) u-PA; Leu144-Leu411) has been constructed and used to analyze promoter activity. Vectors containing the low-M(r) u-PA cDNA coupled to hormone-responsive promoters were introduced to mammalian cells. Following hormone treatment, the activity of the secreted reporter protein was determined in aliquots of cell culture supernatants. The assay is based on plasminogen activation by low-M(r) u-PA and subsequent cleavage of the plasmin-specific tripeptide substrate, S-2251. The resulting chromophore, p-nitroanilide, was quantified colorimetrically at 405 nm. Transient and stable expression of the low-M(r) u-PA reporter gene in different eukaryotic cells demonstrates the suitability of the system for the quantification of the activity of eukaryotic promoter elements in a rapid and highly sensitive manner, while maintaining cell integrity.
已构建编码低分子量（低M(r)）形式的单链尿激酶型纤溶酶原激活剂（低M(r) u-PA；Leu144-Leu411）的报告质粒，并用于分析启动子活性。将含有与激素反应性启动子偶联的低M(r) u-PA cDNA的载体导入哺乳动物细胞。激素处理后，在细胞培养上清液的等分试样中测定分泌的报告蛋白的活性。该测定基于低M(r) u-PA对纤溶酶原的激活以及随后对纤溶酶特异性三肽底物S-2251的切割。产生的发色团对硝基苯胺在405 nm处进行比色定量。低M(r) u-PA报告基因在不同真核细胞中的瞬时和稳定表达表明，该系统适用于以快速且高度灵敏的方式定量真核启动子元件的活性，同时保持细胞完整性。
