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Expression of a Novel Chimeric-Truncated tPA in Pichia pastoris with Improved Biochemical Properties

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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.

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Acknowledgments

The authors are deeply indebted to Dr. Oliver Spadiut (Vienna University of technology, Austria) for his insightful comments. This article is based on part of PhD dissertation and was fully supported by Pasteur Institute of Iran.

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Authors and Affiliations

  1. Biotechnology Research Center, Pasteur Institute of Iran (IPI), No. 69, Pasteur Avenue, Tehran, 1316943551, Iran

    Amirhossein Saadatirad, Soroush Sardari, Mohammadreza Kazemali, Najmeh Zarei, Fatemeh Davami, Farzaneh Barkhordari, Ahmad Adeli & Fereidoun Mahboudi

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  1. Amirhossein Saadatirad
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  2. Soroush Sardari
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  3. Mohammadreza Kazemali
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  4. Najmeh Zarei
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  5. Fatemeh Davami
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  8. Fereidoun Mahboudi
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Correspondence to Fereidoun Mahboudi.

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Saadatirad, A., Sardari, S., Kazemali, M. et al. Expression of a Novel Chimeric-Truncated tPA in Pichia pastoris with Improved Biochemical Properties. Mol Biotechnol 56, 1143–1150 (2014). https://doi.org/10.1007/s12033-014-9794-5

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  • DOI: https://doi.org/10.1007/s12033-014-9794-5

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