title: Expression of a novel chimeric-truncated tPA in Pichia pastoris with improved biochemical properties.
abstract: Thrombolytic therapy by plasminogen activators (PAs) has been a main goal in the treatment of acute myocardial infarction. Despite improved outcomes of currently available thrombolytic therapies, all these agents have different drawbacks that may result in less than optimal outcomes. In order to make tissue plasminogen activator (tPA) more potent, while being more resistant to plasminogen activator inhibitor-1 (PAI-1) and having a higher affinity to fibrin, a new chimeric-truncated form of tPA (CT tPA) was designed and expressed in Pichia pastoris. This novel variant consists of a finger domain of Desmoteplase, an epidermal growth factor (EGF) domain, a kringle 1 (K1) domain, a kringle 2 (K2) domain, in which the lysine binding site (LBS) was deleted, and a protease domain, where the four amino acids lysine 296, arginine 298, arginine 299, and arginine 304 were substituted by aspartic acid. The chimera CT tPA showed 14-fold increase in its activity in the presence of fibrin compared to the absence of fibrin. Furthermore, CT tPA showed about 10-fold more potency than commercially available full-length tPA (Actylase(®)) and provided 1.2-fold greater affinity to fibrin. A residual activity of only 68 % was observed after incubation of Actylase(®) with PAI-1, however, 91 % activity remained for CT tPA. These promising findings suggest that the novel CT tPA variant might be an acceptable PA with superior characteristics and properties.
authors.affiliation: Biotechnology Research Center, Pasteur Institute of Iran (IPI), No. 69, Pasteur Avenue, Tehran, 1316943551, Iran.
authors.forename: Amirhossein
authors.initials: A
authors.lastname: Saadatirad
authors.forename: Soroush
authors.initials: S
authors.lastname: Sardari
authors.forename: Mohammadreza
authors.initials: M
authors.lastname: Kazemali
authors.forename: Najmeh
authors.initials: N
authors.lastname: Zarei
authors.forename: Fatemeh
authors.initials: F
authors.lastname: Davami
authors.forename: Farzaneh
authors.initials: F
authors.lastname: Barkhordari
authors.forename: Ahmad
authors.initials: A
authors.lastname: Adeli
authors.forename: Fereidoun
authors.initials: F
authors.lastname: Mahboudi
mesh_terms: D001761:Bleomycin / Q000494:pharmacology; D005337:Fibrin / Q000378:metabolism; D006801:Humans; D010843:Pichia / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D017395:Plasminogen Activator Inhibitor 1 / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics*; D010959:Tissue Plasminogen Activator / Q000096:biosynthesis* / Q000235:genetics*
chemical_list: D017395:Plasminogen Activator Inhibitor 1; D011993:Recombinant Fusion Proteins; D001761:Bleomycin; C105427:Zeocin; D005337:Fibrin; D010959:Tissue Plasminogen Activator