CONSTRUCTION, EXPRESSION AND CHARACTERIZATION OF TISSUE-TYPE PLASMINOGEN-ACTIVATOR MUTANTS

Three tissue-type plasminogen activator (t-PA) mutants were constructe d by recombinant and site-directed mutagenesis techniques. They are de l(296-302) with deletion of PAI-1 binding site, N117Q/N184Q with degly cosylation of K1 and K2 domains, and their combination mutant designat ed as GGI. Then these three mutants were successfully transiently expr essed in COS-7 cells, and GGI was further stably expressed in CHO cell s. The biological characterization of the expression products indicate d that 'del(296-302) and GGI possessed the resistance to inhibition by PAI-1. In addition, the specific activity of GGI was increased by abo ut 46%, the plasma half-life was prolonged by about one fold, while it s affinity for fibrin was not affected.