Gardell S J, Hare T R, Han J H, Markus H Z, Keech B J, Carty C E, Ellis R W, Schultz L D
Department of Biological Chemistry, Merck Sharp & Dohme Research Laboratories, West Point, Pennsylvania 19486.
Arch Biochem Biophys. 1990 May 1;278(2):467-74. doi: 10.1016/0003-9861(90)90286-8.
The rapidly acting inhibitor of plasminogen activators, PAI-1, was produced intracellularly in Saccharomyces cerevisiae by using the ADH2 promoter to drive the expression of the human PAI-1 cDNA. Approximately 8 mg of human PAI-1 was produced per liter of confluent yeast culture. A purification scheme which resulted in 20% recovery of isolated PAI-1 from the broken yeast cell homogenate was devised. Yeast-derived human PAI-1 differs from endothelial-type PAI-1 isolated from HT1080 fibrosarcoma cells in that the recombinant inhibitor does not contain carbohydrate side chains. Nevertheless, the activity and other functional attributes of yeast-derived PAI-1 are similar to those exhibited by HT1080 fibrosarcoma cell-derived PAI-1. Hence, this study demonstrates that expression of human PAI-1 in yeast is a viable strategy for the production of ample quantities of this key modulator of plasminogen activator-mediated proteolysis.
通过使用ADH2启动子驱动人PAI - 1 cDNA的表达，在酿酒酵母细胞内产生了纤溶酶原激活剂的快速作用抑制剂PAI - 1。每升汇合的酵母培养物可产生约8毫克人PAI - 1。设计了一种纯化方案，可从破碎的酵母细胞匀浆中回收20%的分离PAI - 1。酵母来源的人PAI - 1与从HT1080纤维肉瘤细胞中分离的内皮型PAI - 1不同，在于重组抑制剂不含碳水化合物侧链。然而，酵母来源的PAI - 1的活性和其他功能特性与HT1080纤维肉瘤细胞来源的PAI - 1所表现出的相似。因此，本研究表明在酵母中表达人PAI - 1是生产大量这种纤溶酶原激活剂介导的蛋白水解关键调节剂的可行策略。
