Liu S, Huang P, Huang C
Beijing Institute of Biotechnology, China.
Sci China B. 1995 Nov;38(11):1341-8.
Three tissue-type plasminogen activator (t-PA) mutants were constructed by recombinant and site-directed mutagenesis techniques. They are del(296-302) with deletion of PAI-1 binding site, N117Q/N184Q with deglycosylation of K1 and K2 domains, and their combination mutant designated as GGI. Then these three mutants were successfully transiently expressed in COS-7 cells, and GGI was further stably expressed in CHO cells. The biological characterization of the expression products indicated that del(296-302) and GGI possessed the resistance to inhibition by PAI-1. In addition, the specific activity of GGI was increased by about 46%, the plasma half-life was prolonged by about one fold, while its affinity for fibrin was not affected.
通过重组和定点诱变技术构建了三种组织型纤溶酶原激活剂（t-PA）突变体。它们分别是缺失PAI-1结合位点的del(296 - 302)、K1和K2结构域去糖基化的N117Q/N184Q，以及它们的组合突变体GGI。然后这三种突变体在COS-7细胞中成功实现瞬时表达，GGI在CHO细胞中进一步稳定表达。表达产物的生物学特性表明，del(296 - 302)和GGI具有抵抗PAI-1抑制的能力。此外，GGI的比活性提高了约46%，血浆半衰期延长了约一倍，而其对纤维蛋白的亲和力不受影响。
