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

Autor: Fereidoun Mahboudi, Davami Fatemeh, Barkhordari Farzaneh, Hemayatkar Mahdi, Keivan Majidzadeh-A, Vahid Khalaj, Adeli Ahmad
Přispěvatelé: Biotechnology Research Center, Institut Pasteur d'Iran, Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP), This work was supported by a grant from the Pasteur Institute of Iran, Keivan Majidzadeh-A, Vahid Khalaj, Fatemeh Davami, Mahdi Hemayatkar, Farzaneh Barkhordari, Ahmad Adeli, Fereidoun Mahboudi
Rok vydání: 2010
Předmět:
0106 biological sciences
Protein Folding
[SDV.BIO]Life Sciences [q-bio]/Biotechnology
Plasmin
Gene Expression
Expression
MESH: Pichia
01 natural sciences
Applied Microbiology and Biotechnology
Biochemistry
Tissue plasminogen activator
Pichia
law.invention
MESH: Recombinant Proteins
Pichia pastoris
law
MESH: Tissue Plasminogen Activator
Gene expression
[INFO.INFO-BT]Computer Science [cs]/Biotechnology
Cloning
Molecular
0303 health sciences
Expression vector
biology
Zymography
General Medicine
Recombinant Proteins
Tissue Plasminogen Activator
Recombinant DNA
Human tissue plasminogen activator
Biotechnology
medicine.drug
MESH: Gene Expression
MESH: Protein Folding
Saccharomyces cerevisiae
Bioengineering
03 medical and health sciences
010608 biotechnology
medicine
Humans
MESH: Cloning
Molecular
Molecular Biology
030304 developmental biology
Amidolytic assay
MESH: Humans
T-plasminogen activator
biology.organism_classification
Molecular biology
t-PA
Cloning
Densitometry
Zdroj: Applied Biochemistry and Biotechnology
Applied Biochemistry and Biotechnology, Humana Press, 2010, 162 (7), pp.2037-48. ⟨10.1007/s12010-010-8979-z⟩
ISSN: 1559-0291
0273-2289
DOI: 10.1007/s12010-010-8979-z
Popis: International audience; 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.
Databáze: OpenAIRE
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