Construction, expression and the characterization of t-PA mutants with increased plasma half-life and resistance to inhibition by PAI-1.
Three t-PA mutants, t-PA del K1 (with deletion of K1 domain), t-PA del (296-302) (with deletion of PAI-1 binding site) and their combination mutant t-PA del (K1, 296-302), were constructed by DNA recombination and site-directed mutagenesis techniques. Then the three t-PA mutants were transiently expressed in COS-7 cells, and in addition, the combination mutant t-PA del (K1, 296-302) was stably expressed in CHO cells. The biological analysis of the expression products demonstrated that t-PA del (296-302) and t-PA del (K1, 296-302) had obtained the resistance to inhibition by PAI-1. In addition, the half-life of t-PA del (K1, 296-302) in rat plasma was increased 6 fold while the mutant affinity for fibrin was only slightly affected. Therefore, it was reasonable to consider that the mutant t-PA del (K1, 296-302) may become a potent candidate of new thrombolytic agent.
Liu
S
S
Beijing Institute of Biotechnology, China.
Huang
P
P
Huang
C
C
D000818:Animals; D000911:Antibodies, Monoclonal; D001483:Base Sequence; D016466:CHO Cells / Q000502:physiology; D006224:Cricetinae; D018076:DNA, Complementary; D004591:Electrophoresis, Polyacrylamide Gel; D005337:Fibrin / Q000378:metabolism; D015870:Gene Expression / Q000502:physiology; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D017395:Plasminogen Activator Inhibitor 1 / Q000494:pharmacology*; D017354:Point Mutation; D051381:Rats; D013997:Time Factors; D010959:Tissue Plasminogen Activator / Q000037:antagonists & inhibitors / Q000097:blood / Q000235:genetics*; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D018076:DNA, Complementary; D017395:Plasminogen Activator Inhibitor 1; D005337:Fibrin; D010959:Tissue Plasminogen Activator