earticle

영어

Staphylokinase and its variant (Staphylokinase-Hirulog), the third generation thrombolytic molecule plays an significant role in treating the acute myocardial infarction and stroke which are leading causes of morbidity and mortality across the globe. In refolding of the staphylokinase and its variant from industrially feasible pET28a+, gradual decrease in concentration of urea was used to solubilise the protein. But urea is a chaotropic denaturant, which unravel the structure of proteins by destabilizing internal, non-covalent bonds between atoms. In this study, we selected the expression system to have the increased soluble fraction of sak and its variant from IPTG inducible expression host Escherichia coli BL21(DE3). The expression level and solubility of recombinant sak and its variant from cold shock expression vector pCOLDI was induced at the low temperature (15°C) was highly contrast to the induction process at 37 °C from pET28a+, which gives insoluble fractions. Recombinant sak and its variant were expressed primarily as soluble protein using pCOLDI, later purified with Nickel-chelating resin (Ni-NTA) and the quantity of the purified protein was 913 mg/L. Soluble fractions of purified sak variant having the fibrinolytic activity of 21,825 U/ml and specific anti-thrombin activity of 1,200 ATU/mg. For the first time soluble fractions was achieved instead of inclusion bodies without compromising the quantity and activity of the protein and working with urea was excluded.