title: Engineering, expression and purification of a chimeric fibrin-specific streptokinase.
abstract: Streptokinase is a valuable fibrinolytic agent used to cope with myocardial infarction and brain stroke. Despite its high efficiency in dissolving blood clots, streptokinase (SK) has no specificity in binding fibrin, causing some problems such as internal bleedings following its administration. To make streptokinase fibrin specific and limit the fibrinolytic process to the clot location, we engineered a chimeric streptokinase by fusing the fibrin binding Kringle 2 domain of tissue plasminogen activator (TPA) to the streptokinase N-terminal end. The chimeric SK construct (KSK) with inserted Kringle 2 domain was cloned into pET28a expression vector. The expression of recombinant protein was carried out in Escherichia coli origami (DE3) and confirmed by SDS-PAGE and Western blotting analyses. We used the chromogenic substrate S-2251 method to assess the specific activities of the chimeric and control wild-type proteins. Then, the two proteins were added in amounts with equal activity to fibrin clots of identical size. Finally, the supernatant above the fibrin clots was collected and subjected to the chromogenic assay to analyze the specificity of the chimeric protein. The specific activities of the chimeric and wild-type proteins were found to be 0.06 U/mg and 0.07 U/mg, respectively. Because of the binding of the chimeric protein to fibrin, the mean specific activity was significantly lower in the KSK supernatant (0.01) compared with the control (approximately 0.06) (p < 0.05). Our in vitro results indicate that the chimeric streptokinase protein has strong fibrin-specific activity compared to the wild-type protein. However, further in vivo studies are needed to evaluate its potential fibrinolytic effects.
authors: Department of Medical Biotechnology, School of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran; Student Research Committee, Shiraz University of Medical Sciences, Shiraz, Iran; Diagnostic Laboratory Sciences and Technology Research Center, School of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran.
authors: Mohammad Naser
authors: MN
authors: Taheri
authors: Department of Medical Biotechnology, School of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran; Diagnostic Laboratory Sciences and Technology Research Center, School of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran.
authors: Abbas
authors: A
authors: Behzad-Behbahani
authors: Department of Medical Biotechnology, School of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran; Diagnostic Laboratory Sciences and Technology Research Center, School of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran.
authors: Gholamreza
authors: G
authors: Rafiei Dehbidi
authors: Department of Medical Biotechnology, School of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran; Student Research Committee, Shiraz University of Medical Sciences, Shiraz, Iran; Diagnostic Laboratory Sciences and Technology Research Center, School of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran.
authors: Saeede
authors: S
authors: Salehi
authors: Department of Medical Biotechnology, School of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran; Diagnostic Laboratory Sciences and Technology Research Center, School of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran. Electronic address: sharifsd@suma.ac.ir.
authors: Sedigheh
authors: S
authors: Sharifzadeh
mesh_terms: D001426:Bacterial Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005337:Fibrin / Q000737:chemistry / Q000378:metabolism; D005342:Fibrinolysis; D006801:Humans; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D013291:Streptococcus / Q000201:enzymology / Q000235:genetics*; D013300:Streptokinase / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D010959:Tissue Plasminogen Activator / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
chemical_list: D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; D005337:Fibrin; D013300:Streptokinase; D010959:Tissue Plasminogen Activator
keywords: Chromogenic substrate assay; Fibrin-binding streptokinase; Fibrinolytic agent; Kringle 2