[Expression and activity of tissue-type plasminogen activator mutant reteplase with deletion of PAI-1 binding sites].
OBJECTIVE
To construct a mutant of human tissue plasminogen activator (t-PA) with activity not inhibited by plasminogen activator inhibitor-1 (PAI-1).


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
Zhu
Mei-cai
MC
Research Center of Clinical Molecular Biology, Air Force General Hospital, Beijing 100036, China. zhumc@yahoo.com
Zhan
Zhi
Z
Wang
Ya-jing
YJ
Liu
Cheng-gang
CG
Shi
Ying
Y
Cai
Qing
Q
D001665:Binding Sites / Q000235:genetics; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D017395:Plasminogen Activator Inhibitor 1 / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011485:Protein Binding / Q000235:genetics; D010959:Tissue Plasminogen Activator / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*
D017395:Plasminogen Activator Inhibitor 1; D010959:Tissue Plasminogen Activator