Expression Of Recombinant Targeting Bifunctional Hirudin In Pichia Pastoris

Hirudin is an anticoagulative protein with a molecular weight of 7,000Da secreted from salivary glands of Hirudo medicinalis. The naturally occurring hirudin is known as the most potent and specific inhibitor of thrombin, can bind directly to thrombin, inhibit fibrin-bound as well as fluid-phase thrombin. It has high affinity with thrombin to form a reversible complex with 1:1 non-covalent bonding. As a drug to prevent thrombus, it has various advantages, like the stable and sustained effect to specifically inhibit the activity of thrombin directly with rare side effects; in addition, it has teeny antigenicity and little toxicity for its low molecular weight.Many problems hinder the clinical application of Hirudin. First of all, Hirudin would prolong the bleeding time, which can induce the bleeding of thrombolysis position, while inhibit the activity of thrombin. Even worse, there are rare antagonists of bleeding induced by Hirudin. Furthermore, Hirudin can only inhibit the activity of thrombin, which is effective in phlebothrombosis but not arteriothromobosis.In order to decrease the side effects of Hirudin and increase its activity to prevent arteriothromobosis, we fused a FXa recognition sequence into N'of Hirudin to decrease the effect of bleeding through elevating of its targeting, while maintaining the activity of natural Hirudin. In addition, we fused Arg-Gly-Asp (RGD) sequence into appropriate genetic locus of Hirudin to elevate its activity of inhibiting platelet aggregation to increase its activity of preventing arteriothromobosis. Furthermore, we added a 9×His-Tag to make its separation and purification conveniently, which provided an efficient methed to produce a great deal of fusion Hirudin protein.In this study, the recombination Hirudin gene was successfully cloned by PCR with 6 primers designed according to the amino acid sequence of our fusion protein and the codon preference of Pichia pastoris, and an expression vector was constructed by ligating the recombination Hirudin gene into the Pichia pastoris vector pPIC9K, which was transfered into Pichia pastoris GS115. Then a positive clone, which was identified as Mut+, was obtained with G418 selection, and expressed efficiently, stably and secretely in suitable conditions.The conditioned medium was concentrated with Millipore ultrafiltration and the interest protein was purified by affinity chromatography. Finally, with tryptic digestion, western blotting, and thrombin titration, we found that the activity of inhibiting thrombin of our fusion protein and the natural Hirudin has no difference.