Construction, expression and refolding of a bifunctional fusion protein consisting of C-terminal 12-residue of hirudin-PA and reteplase.

To obtain a bifunctional protein simultaneously showing bioactivity of anticoagulant and fibrinolytic for use in the treatment of thrombotic diseases, we constructed a fusion protein (HV12p-rPA) containing C-terminal 12-residue of hirudin-PA (HV12p) and reteplase (rPA). The fusion protein, in which HV12p was linked to rPA via Gly-Gly-Gly, was successfully expressed in an inactive form of inclusion bodies in Escherichia coli. HV12p-rPA was identified by sodium dodecylsulfate-polyacrylamide gel electrophoresis. The expression level of HV12p-rPA was optimized by an orthogonal method and finally enhanced from 12 % to approximate 30 %. We also deeply investigated the condition of renaturation of HV12p-rPA, and the inactive protein was partly renatured through various conditions. The refolding efficacy of HV12p-rPA estimated by the recovery of fibrinolytic activity varied from 0.03 % to 16.6 % and the anticoagulant activity fluctuated in the range from 41 to 2,297 ATU/mg. Bioassays indicated that the resulted fusion protein, as expected, exhibited both fibrinolytic and anticoagulant activities. These works laid a foundation for further characterization of HV12p-rPA.