Cloning and purification of an anti-thrombotic, chimeric Staphylokinase in Pichia pastoris

There has been an increasing prevalence of cardiovascular diseases such as myocardial infarction and stroke in modern societies because of multiple lifestyle related issues like sedentariness and obesity, alcohol consumption and many more "life-style"factors. The FDA-approved thrombolytics such as Tissue Plasminogen Activator, Streptokinase etc. are used to lyse the clots in thrombotic disorders such as myocardial infarction, stroke etc. but re-occlusion and bleeding that are co-incident to their clinical usage are not addressed. Hence, there is need to develop thrombolytics having properties like increased fibrin clot specificity and thrombin inhibition capability to prevent re-occlusion. In the present work, a fusion protein construct containing two components i.e. Staphylokinase (SAK) and Epidermal Growth Factor (EGF) 4, 5, 6-like domains of human thrombomodulin (THBD) was expressed in Pichia pastoris after genetic optimization. SAK isolated from Staphylococcus aureus is a fibrin-specific plasminogen activator while EGF 4, 5, 6-like domains are reported to be responsible for imparting thrombin inhibition to human thrombomodulin, and therefore, expected could help prevent re-occlusion in the novel construct - SAK_EGF, which is a 43 kDa protein. After expression, it was purified (approx. 13-fold) using two-step purification protocol involving ion-exchange followed by Gel Filtration Chromatography (GFC). The functional characterization including plasminogen activation and thrombin inhibition showed that both the fusion partners viz. SAK and 4,5,6 EGF-like domains retained their respective activities after fusion, confirming it to be a bio-active construct. Thus, this engineered protein could be clinically promising due to the combinatorial effect of fibrin-specific thrombus lysis and prevention of re-occulusion.