The Effect of Signal Sequence and His-tags on the Expression and Anti-Thrombin Activity of Recombinant Hirudin

Background Adding signal sequence and His- tags to the expression vectors of recombinant proteins usually increased the production yield by promoting protein secretion to the periplasmic space and by facilitating the purification processes.In this study, three different expression constructs including vectors with or without signal sequence and His-tags were designed to compare the effect of these elements on Hirudin expression and function. Hirudin is a natural anticoagulant protein produced in the salivary glands of leeches.MethodsHirudin variants including cytoplasmic Hirudinn without His-tag (cr-Hirudin),  cytoplasmic Hirudin with His-tag (crhis-Hirudin), and periplasmic Hirudin with His-tag (prhis-Hirudin) constructs were expressed in E.coli BL 21(DE3). Ion exchange chromatography and ion metal affinity chromatography were applied for protein purification. The prothrombin time (PT) and activated partial thromboplastin time (aPTT) assays were performed to assess the anti-thrombin bioactivity of variants.Results The expression rate of prhis-Hirudin was approximately 1.6 fold higher than cytoplasmic variants ( cr-Hirudin and crhis-Hirudin). The prothrombin time and activated partial thromboplastin time of crhis-Hirudin and prhis-Hirudin were similar and approximately 20 and 55 % lower than those of cr-Hirudin.Conclusion Applying signal peptide or His-tag increased the production yield of recombinant hirudin but had negative contributions to its activity.