[Expression and activity of tissue-type plasminogen activator mutant reteplase with deletion of PAI-1 binding sites]

To construct a mutant of human tissue plasminogen activator (t-PA) with activity not inhibited by plasminogen activator inhibitor-1 (PAI-1).

The finger, epidermal growth factor and kringle-1 domains were removed from native t-PA to obtain a t-PA mutant with the PAI-1 binding sites. A pUC18 plasmid containing the human t-PA full-length cDNA sequence was used as template, and the DNA sequences encoding 1-3 and 176-527 amino acids were amplified by PCR. The nucleotides AAG CAC AGG AGG (from 373 to 384) in the PAI-1 binding site changed to GCG GCC GCG GCG, so amino acid KHRR was replaced by AAAA correspondently.

Sequencing result showed that the above t-PA mutant DNA sequence was consistent with the expected. Then it was cloned in an E.coli expression vector and was highly expressed. The expressed protein occupied about 30% of total bacterial proteins as inclusion body. After denaturation and renaturation, the active t-PA mutant was obtained,activity of which was not inhibited by PAI-1.

This t-PA mutant may be developed to a new gene-engineering drug with higher therapeutic activity and lower dose requirement in the treatment of acute myocardial infarction and cerebrovascular thrombosis diseases.