Designing a multifunctional staphylokinase variant (SAK-2RGD-TTI) with appropriate thrombolytic activity in vitro
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Thrombin, platelets, and plasmin are three key factors involved in hemostasis and thrombolysis. Thrombolytic therapy with clinically approved drugs is often followed by recurrent thrombosis caused by thrombin-induced platelet aggregation from the clot debris. In order to minimize these problems, new constructs were designed for the expression of recombinant staphylokinase (rSAK) and also a fusion protein composed of staphylokinase, 20 amino acids containing 2 RGD followed by tsetse thrombin Inhibitor (SAK-2RGD-TTI) in Pichia pastoris.
Modeling the tertiary structure of SAK-2RGD-TTI showed that the linker containing RGD and TTI did not interfere with proper folding of SAK. In laboratory testing, the purified SAK-2RGD-TTI (420 μg/mL) dissolved an average of 45% of the blood clot. The activity of the SAK-2RGD-TTI was also confirmed in various tests including human plasminogen activation assay, fibrin clot lysis assay, well diffusion method, activated partial thromboplastin time and platelet rich clot lysis assay.
Our findings suggest that SAK-2RGD-TTI has improved therapeutic properties preventing reocclussion. It further confirms that it is practicable to assemble and produce a hybrid multifunctional protein that targets hemostatic process at various stages.
KeywordsProcess engineering Staphylokinase Tsetse thrombin inhibitor peptide Yeast
We appreciated Dr. Mohammad Soukhtanloo, Dr. Mostafa Khedri and Dr. Manouchehr Teymouri for their valuable comments.
Supplementary Figure 1—The secondary structure of the SAK-2RGD-TTI protein predicted by SOPMA online software. Amino acid residues of N-terminal, which is indeed a catalytic site for fibrinolytic activity, has more the random coil structure.
Supplementary Figure 2—Ramachandran plot analysis of the SAK-2RGD-TTI using the RAMPAGE online software. It is based on torsional angles (Phi or ф and Psi or ψ) of amino acid residues (A) led to their falling in energetically favoured, allowed or outlier regions. More amino acids in favoured and allowed regions, the model more valid. The conformations and location of amino acids is shown in four individual plots having heading as general (all amino acids except Glycine, Proline and Pre-Pro (amino acid before proline), Glycine, Pre-Pro and Proline. Amino acids marked with red rectangleplace in outlier region.
This study was supported by the Pharmaceutical Research Center, Buali (Avicenna) Research Institute, Mashhad University of Medical Sciences with Grant No. 921731.
Compliance with ethical standards
Conflict of interest
The authors have declared no conflict of interest with the current work or its publication.
All procedures were performed in accordance with the ethical standards of the local ethics committee of Mashhad University of Medical Sciences (Iran) and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards (Grant No: 921731).
All authors are aware of and agree to the content of the manuscript.
Research involving human participants and/or animals
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