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.

Combined TGE-SGE Expression of Novel PAI-1-Resistant t-PA in CHO DG44 Cells Using Orbitally Shaking Disposable Bioreactors

“…Chinese Hamster Ovary (CHO) cells are the major mammalian host for the production of recombinant proteins that are both compatible and bioactive in humans. The production of t-PA in E. coli, Leishmania tarentolae, and Pichia pastoris was previously tested by the current authors [11,16,26]. However, active t-PA was poorly expressed in E. coli (3-7 IU/ml) [21] owing to a lack of posttranslational modifications in this host.…”

Structural Biology and Protein Engineering of Thrombolytics

“…Higher yields of properly folded t-PA were achieved via expression into the periplasm or co-expression with disulfide oxidoreductase [361,362]. Production in yeasts generally leads to higher yields and absence of endotoxin when compared to bacteria [[363], [364], [365]]. Other avenues are auto-induction, growth medium or cultivation protocol optimization [366,367], development of new refolding protocols [[368], [369], [370]] or new mammalian cell lines [371].…”

The evolution of recombinant thrombolytics: Current status and future directions

“…67 An efficient, properly processed and glycosylated form of recombinant t-PA (rt-PA) was produced using mammalian cells with properties similar to the Bowes cell line t-PA. 68 Majidzadeh-A and co-workers (2010) expressed the gene encoding full-length human tPA under the control of the AOX1 promoter and S. cerevisiae a-mating factor signal sequence in P. pastoris GS115 with an amidolytic activity of 1,650 IU/ml. 69 Human t-PA (located on chromosome 8) was first isolated as a 70 kDa (527 amino acids) single chain polypeptide that is converted to the active 2-chain form via plasmin-mediated cleavage across the Arg 275 -Ile 276 bond. 43,67 Unlike scuPA, single-chain tPA has significant enzymatic activity and is not a true zymogen.…”