Skip to Main content Skip to Navigation
Journal articles

Cloning and expression of functional full-length human tissue plasminogen activator in Pichia pastoris.

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.
Document type :
Journal articles
Complete list of metadatas

Cited literature [41 references]  Display  Hide  Download

https://hal-riip.archives-ouvertes.fr/pasteur-00763506
Contributor : Fatemeh Davami <>
Submitted on : Tuesday, December 11, 2012 - 8:13:27 AM
Last modification on : Wednesday, September 23, 2020 - 10:48:02 AM
Long-term archiving on: : Sunday, December 18, 2016 - 12:03:18 AM

File

 Restricted access
To satisfy the distribution rights of the publisher, the document is embargoed until : jamais

Please log in to resquest access to the document

Identifiers

Collections

Citation

Keivan Majidzadeh-A, Vahid Khalaj, Davami Fatemeh, Hemayatkar Mahdi, Barkhordari Farzaneh, et al.. Cloning and expression of functional full-length human tissue plasminogen activator in Pichia pastoris.. Applied Biochemistry and Biotechnology, Humana Press, 2010, 162 (7), pp.2037-48. ⟨10.1007/s12010-010-8979-z⟩. ⟨pasteur-00763506⟩

Share

Metrics

Record views

134