Expression, purification and characterization of recombinant targeting bifunctional hirudin in Pichia pastoris

Abstract

A recombinant targeting bifunctional hirudin was expressed in the yeast Pichia pastoris. In order to decrease the side effects of hirudin and increase its activity to prevent arterial thrombus, we fused a factor Xa (FXa) recognition sequence into N’ of hirudin, while maintaining the activity of natural hirudin. In addition, an Arg-Gly-Asp (RGD) sequence was fused into appropriate genetic locus of hirudin. Furthermore, we added a 9 × His - Tag to make its separation and purification conveniently. The recombination hirudin gene was successfully cloned and ligated into the P. pastoris vector pPIC9K to form an expression vector, which was transferred into P. pastoris GS115. A transformant strain was selected and expressed efficiently in suitable conditions. Then, the fusion protein was purified by affinity chromatography. Through anti-thrombin activity analysis and anti-platelet aggregation activity analysis, we found that the anti-thrombin activity of the fusion protein did not
change comparing with the natural hirudin, whereas its anti-platelet capability was enhanced.