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. 2012 Jul;4(3):142-7.

Development of an immunoaffinity method for purification of streptokinase

Zohreh Karimi  1 , Mohammad Babashamsi, Ezat Asgarani, Ali Salimi
Affiliations

Development of an immunoaffinity method for purification of streptokinase

Zohreh Karimi et al. Avicenna J Med Biotechnol. 2012 Jul.

Abstract

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-Activated 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.

Keywords: Chromatography; Purification; Streptokinase; Thrombolytic agent.

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Figures

Figure 1
Figure 1
10% SDS-PAGE; fusion GST-SK expression in E.coli, BL21 PLYsS: Lane 1: Pre-induction. Lane 3: 2 hr, Lane 4: 4 hr, Lane 5: 6 hr and Lane 6: 8 hr post-induction. Lane7: Protein marker (Fermentas SM0441). Lane 8: Pure SK
Figure 2
Figure 2
Western Blot of fusion GST-SK expression in E.coli, BL21 PLYsS: Lane 1: Protein MW marker (Fermentas SM0671). Lanes 2, 3, 4, 5 and 6: Cloned GST-SK in E.coli, after induction. Lane 7: BSA. Lanes 8, 9 and 10: Pure SK
Figure 3
Figure 3
10% SDS-PAGE; purification of anti-SK from rabbit sera, Lane 1: BSA, Lane 2: Flow-through, Lane 3: serum, Lanes 4, 6, and 7: Pure anti-SK, Lane 8: IgG
Figure 4
Figure 4
10% SDS-PAGE; purification of SK from H46a, by immunoaffinity column Lane 1: Cultured H46a, Lane 2: Flowthrough, Lane 3: Protein marker, Lane 4 and 5: Purified SK, Lane 6: BSA
Figure 5
Figure 5
10% SDS-PAGE; purification of recombinant fusion GST-SK from E.coli, by immunoaffinity column. Lane 1: Purified GST-SK 73 kD, Lane 2: Flow-through, Lane 3: Sonicate of E.coli, Lane 4: Protein marker (Fermentas SM0441), Lane 5: Hand made marker (Reduced IgG and BSA), Lane 6: BSA, Lanes 7 and 8: Non-transformed E.coli
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