A prourokinase-RGDS chimera - Construction, expression and characterization

A tetrapeptide, RGDS, was inserted into proUK kringle domain G118-L119 by t he construction of a mutant proUK-RGDS gene. The gene was expressed in the baculovirus expression system. Immunoaffinity chromatography was used to pu rify the chimera and protein with purity over 90% was achieved. The chimera was tested for its platelet membrane binding function and showed a calcium -dependent platelet binding activity. Amidolytic activity of the chimera wa s tested. The result indicated that specific amidolytic activity of plasmin activated chimera was 62 000 IU/mg, comparable to the previously reported 65 355 IU/mg of plasmin activated natural proUK([1]). Activation of plasmin ogen by the chimera after plasmin treatment followed Michieal-Menten kineti cs, and the Km was 0.97 mu mol/L, which was also comparable to 1.64 mu mol/ L of native urokinase. The chimera also showed intensive ability to inhibit platelet aggregation in vitro. These results indicate that this chimera mi ght be useful as a bifunctional thrombolytic agent.