The construction and expression of deletion mutant of human pro-urokinase cDNA.
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.
Han
S
S
Institute of Biotechnology, Academy of Military Medical Sciences, Beijing, China.
Yu
W
W
Cheng
D
D
Hu
B
B
D000818:Animals; D016466:CHO Cells; D019556:COS Cells; D003001:Cloning, Molecular; D006224:Cricetinae; D018076:DNA, Complementary; D015870:Gene Expression; D006801:Humans; D010957:Plasmids; D011498:Protein Precursors / Q000032:analysis / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000235:genetics; D017384:Sequence Deletion; D014568:Urokinase-Type Plasminogen Activator / Q000032:analysis / Q000235:genetics*
D018076:DNA, Complementary; D011498:Protein Precursors; D011993:Recombinant Fusion Proteins; D014568:Urokinase-Type Plasminogen Activator