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Cloning and expression of a new recombinant thrombolytic and anthithrombotic agent - a staphylokinase variant

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Kowalski M., Brown G., Bieniasz M., Oszajca K., Chabielska E., Pietras T., Szemraj Z., Makandjou-Ola E., Bartkowiak J., Szemraj J.

issue 1

41-53

To develop a more potent antithrombin agent with thrombolytic and antiplatelet properties, a new staphylokinase (SAK) variant was constructed. The kringle 2 domain (K2) of tissue type-plasminogen activator (t-PA) containing a fibrin-specific binding site (i), the RGD sequence (Arg-Gly-Asp) for the prevention of platelet aggregation (ii) and the antithrombotic agent - hirulog (iii) was assembled to the C-terminal part of recombinant staphylokinase (r-SAK). cDNA for the hybrid protein SAK-RGD-K2-Hirul was cloned into Pichia pastoris pPIC9K yeast expression vector. The introduction of K2 t-PA, the RGD sequence and hirulog into the C-terminus of r-SAK did not alter the staphylokinase activity. We observed a higher clot lysis potency of SAK-RGD-K2-Hirul as evidenced by a faster and more profound lysis of 125I-labeled human fibrin clots. The potency of thrombin inhibition by the hirulog C-terminal part of the recombinant fusion protein was almost identical to that of r-Hir alone. These results suggest that the SAK-RGD-K2-Hirul construct can be a more potent and faster-acting thrombolytic agent with better antithrombin and antiplatelet properties compared to r-SAK and SAK-RGD-K2-Hir.

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1 Acta Biochimica Polonica

1 2009

1 Bartkowiak J.

1 Bieniasz M.

1 Brown G.

1 Chabielska E.

1 Kowalski M.

1 Makandjou-Ola E.

1 Oszajca K.

1 Pietras T.

1 Szemraj J.

1 Szemraj Z.

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Cloning and expression of a new recombinant thrombolytic and anthithrombotic agent - a staphylokinase variant