Construction, expression and characterization of tissue-type plasminogen activator mutants.

Abstract

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.