Repository of Research and Investigative Information

Repository of Research and Investigative Information

Hormozgan University of Medical Sciences

Comparison of Expression Optimization of New Derivative of Staphylokinase (SAK-2RGD-TTI) with the rSAK.

(2019) Comparison of Expression Optimization of New Derivative of Staphylokinase (SAK-2RGD-TTI) with the rSAK. Biotechnol Prog.

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Staphylokinase (SAK) is a promising thrombolytic agent for the treatment of patients suffering from blood-clotting disorders. To increase the potency of SAK and to minimize vessel reocclusion, a new construct bearing SAK motif fused to tsetse thrombin inhibitor (TTI) via a 20-amino acid linker with 2 RGD (2 x Arginine-Glycine-Aspartic acid inhibiting platelet aggregation via attachment to integrin receptors of platelet) was codon optimized and expressed comparatively in Pichia pastoris GS115 as a Mut+ strain and KM71H as a Muts strain. Fusion protein was optimized in terms of best expression condition and fibrinolytic activity and compared with the rSAK. Expression level of the designed construct reached up to 175 mg/L of the culture medium after 72-hr stimulation with 2.5% methanol and remained steady for 3-4 days. The highest expression was obtained at the range of 2-3% methanol. The SAK-2RGD-TT (relative activity > 82%) was more active at 25-37°C than rSAK (relative activity of 93%). Further, it showed relative activity > 80% at pH ranges of 7-9. Western blot analysis showed two bands of nearly 27 and 24 kDa at ratio of 5 to 3, respectively. The specific fibrinolytic activity of the SAK-2RGD-TTI was measured as 8269 U/mg, and 19616 U/mg for the non-purified and purified proteins, respectively. Deglycosylation by using tunicamycin in culture medium resulted in higher fibrinolytic activity of SAK-2RGD-TTI (2.2 fold). Consequently, compared to the rSAK, at the same equimolar proportion, addition of RGD and TTI fragments could increase fibrinolytic activity. Also, P. pastoris can be considered as an efficient host for overexpression of the soluble SAK-2RGD-TTI with high activity without requiring a complicated purification procedure. This article is protected by copyright. All rights reserved.

Item Type: Article
Keywords: Anti platelet aggregation; Antithrombin; Codon optimization; Pichia pastoris; Staphylokinase
Subjects: QW Microbiology and Immunology > QW 1-300 Microbiology
Depositing User: هدی فهیم پور

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