Development of thermostable Streptokinase by recombinant DNA technology as fibrinolytic agent for different thromboembolic disorders

Background: Edicine is a therapeutic agent in the management of thromboembolic blockade. Streptokinase is presently utilized in clinical mase and is an extracellular protein secreted by certain strains of beta-hemolytic streptococci. It activates plasminogen to produce plasmin, an enzyme that breaks down fibrin clots through specific lysine binding sites. Purpose of the research: Production of thermostable streptokinase by recombinant DNA technology as a fibrinolytic agent for various thromboembolic diseases. This study aimed to improve the physicochemical properties of the streptokinase enzyme to increase its thermal stability and exhibit superior fibrinolytic activity against various thromboembolic diseases. Research type: Screening experimental study. Our study type was to screen the thermostability and biological activity of modified streptokinase generated by bioinformatics. Methodology: Isolation of SPP-producing streptokinase was performed on blood agar. Streptococcus agalactiae was the main isolate in screening studies. In this work, streptokinase was generated by bioinformatic recombinant DNA techniques by placing cysteine-cysteine side by side in the core alpha-helix of this fibrinolytic enzyme. The streptokinase product was pre-extracted by salt precipitation with ammonium sulfate and then purified by affinity chromatography. A casein digestion method was used to detect and determine the biological activity of test enzymes compared to standard streptokinase enzymes. The expression host was Pichia pastoris SMD1168. The C-terminus is 6x histidine, the inducer is methanol, the promoter is AUG1, and PYES2-DEST52 is an expression vector. Result: This research led to improvements in the physicochemical properties of the streptokinase enzyme, making it a thermostable drug. The test enzyme has proven to be a good fibrinolytic agent in thromboembolic stroke, pulmonary and cardiac thrombosis. In addition, it was thermally stable enough to be stored outside the refrigerator (2-8°C) at room temperature. The standard enzyme is a heat-labile drug and could not be stored at room temperature. The yield of recombinant streptokinase was approximately 470 mg/L of the initial culture.Conclusion: Thermostable streptokinase protein produced by recombinant DNA technology, showed high efficacy as a thrombolytic agent for the dissolution of various thromboembolic disorders.