Construction, expression and characterization of tissue-type plasminogen activator mutants.
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.
Liu
S
S
Beijing Institute of Biotechnology, China.
Huang
P
P
Huang
C
C
D000818:Animals; D016466:CHO Cells / Q000378:metabolism; D006224:Cricetinae; D018076:DNA, Complementary / Q000235:genetics; D006801:Humans; D008657:Metabolic Clearance Rate; D016297:Mutagenesis, Site-Directed; D017395:Plasminogen Activator Inhibitor 1 / Q000494:pharmacology; D017384:Sequence Deletion; D010959:Tissue Plasminogen Activator / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics*; D014162:Transfection
D018076:DNA, Complementary; D017395:Plasminogen Activator Inhibitor 1; D010959:Tissue Plasminogen Activator