Han S, Yu W, Cheng D, Hu B
Institute of Biotechnology, Academy of Military Medical Sciences, Beijing, China.
Chin J Biotechnol. 1997;13(2):71-7.
The human pro-urokinase mutant deleting 150-156 amino acids was constructed by overlap-extension PCR and other molecular cloning techniques. The mutant was expressed transiently in COS-7 cells and constitutively in CHO cells; the expression level is 450-500 IU/10(6) cells/24 h. SDS-PAGE and Western blotting analysis showed that the molecular weight of the expression product is 54 kDa, which is similar to that of the mature pro-UK and recombinant pro-UK (rPro-UK) expressed by full-length cDNA. Most of the products exist in the form of single chain; the percentage of single chain is much higher than that of rPro-UK. In addition, the mutant product is more resistant to proteinases and its affinity to fibrin is also improved slightly.
通过重叠延伸PCR和其他分子克隆技术构建了缺失150 - 156个氨基酸的人尿激酶原突变体。该突变体在COS - 7细胞中瞬时表达，在CHO细胞中组成性表达；表达水平为450 - 500 IU/10(6)细胞/24 h。SDS - PAGE和Western印迹分析表明，表达产物的分子量为54 kDa，与由全长cDNA表达的成熟尿激酶原和重组尿激酶原（rPro - UK）相似。大部分产物以单链形式存在；单链的比例远高于rPro - UK。此外，突变体产物对蛋白酶更具抗性，其对纤维蛋白的亲和力也略有提高。
