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Applied Biochemistry and Biotechnology

, Volume 162, Issue 7, pp 2037–2048 | Cite as

Cloning and Expression of Functional Full-Length Human Tissue Plasminogen Activator in Pichia pastoris

  • Keivan Majidzadeh-A
  • Vahid Khalaj
  • Davami Fatemeh
  • Hemayatkar Mahdi
  • Barkhordari Farzaneh
  • Adeli Ahmad
  • Fereidoun MahboudiEmail author
Article

Abstract

Human tissue plasminogen activator (t-PA) plays a pivotal role in the treatment of acute myocardial infarction, ischemic stroke, and deep vein thrombosis. It has the benefit of generating no adverse effects such as fibrinogen depletion, systemic hemorrhage, and immunologic reactions. Human t-PA is a serine-protease enzyme containing 527 amino acid residues in five structural domains. The correct folding of t-PA requires the correct pairing of 17 disulfide bridges in the molecule. A gene encoding full-length human t-PA was cloned into pPICZαA expression vector downstream of alcohol oxidase promoter and α-mating signal sequence from Saccharomyces cerevisiae and flush with the kex2 cleavage site to express the protein with a native N terminus. The methylotrophic yeast, Pichia pastoris GS115 strain, was transformed with this cassette, and methanol utilizing (mut+) transformants were selected for production and secretion of human t-PA into culture media. SDS–PAGE and Western blot analysis showed the expressed bands of t-PA protein. Zymography test indicated suitable folding and proper function of the expressed recombinant human t-PA in conversion of plasminogen to plasmin and gelatin lysis. Amidolytic activity test showed the amidolytic activity of 1,650 IU/ml. The results of this study concluded that P. pastoris methylotrophic yeast can be a suitable alternative for mammalian and prokaryotic expression systems to produce t-PA.

Keywords

Human tissue plasminogen activator t-PA Pichia pastoris Cloning Expression Zymography Amidolytic assay Densitometry 

Notes

Acknowledgments

This work was supported by a grant from the Pasteur Institute of Iran. We thank Dr. Mohammad Soleimani for his collaboration in this study. We also thank Dr. Jonathan Roger Beckwith from Harvard University and Dr. George Georgiou from University of Texas for commenting on this manuscript.

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Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Keivan Majidzadeh-A
    • 1
  • Vahid Khalaj
    • 1
  • Davami Fatemeh
    • 1
  • Hemayatkar Mahdi
    • 1
  • Barkhordari Farzaneh
    • 1
  • Adeli Ahmad
    • 1
  • Fereidoun Mahboudi
    • 1
    Email author
  1. 1.Biotechnology DepartmentPasteur Institute of IranTehranIran

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