Development of an Immunoaffinity Method for Purification of Streptokinase

Background: streptokinase is a potent activator of plasminogen to plasmin, the enzyme that can solubilize the fibrin network in blood clots. streptokinase is currently used in clinical medicine as a thrombolytic agent. it is naturally secreted by β-hemolytic streptococci. methods: to reach an efficient method of purification, an immunoaffinity chromatography method was developed that could purify the streptokinase in a single step with high yield. at the first stage, a cnbr-ac-tivated sepharose 4b-lysine column was made to purify the human blood plasminogen. the purified plasminogen was utilized to construct a column that could purify the streptokinase. the rabbit was immunized with the purified streptokinase and the anti-streptokinase (igg) purified on another streptokinase substituted sepharose-4b column. the immunoaffinity column was developed by coupling the purified anti-streptokinase (igg) to sepharose 6mb–protein a. the escherichia coli (e.coli) bl21 (de3) plyss strain was transformed by the recombinant construct (cloned streptokinase gene in pgex-4t-2 vector) and gene expression was induced by iptg. the expressed protein was purified by immunoaffinity chromatography in a single step. results: the immunoaffinity column could purify the recombinant fusion gst-sk to homogeneity. the purity of streptokinase was confirmed by sds-page as a single band of about 71 kd and its biological activity determined in a specific streptokinase assay. the yield of the purification was about 94%. conclusion: this method of streptokinase purification is superior to the previous conventional methods