Hirudin display on the surface of bacteriophage M13

Hirudin was fused to the N terminus of M13 minor protein gp3 (197-406) through a linker GGGS by inserting both the hirudin gene and the gp8 signal sequence into the modified phagemid vector pCANTAB 5V to construct pCANTAB 5G8-Hir. The expressed fusion protein was directed by gp8 signal peptide into the periplasm and assembled to the phage particle to form the hirudin-phage. The fusion protein and fusion phage were detected with biotin-thrombin by Western blotting analysis. Antithrombin activity analysis confirmed that the hirudin portion in the fusion protein and fusion phage bear similar native conformation. The successful display of hirudin on the surface of M13 phage laid a sound foundation for the further study on directed evolution of antithrombotic proteins with altered properties.