Expression And Secretion Of The Recombinant UKAV And Its Biological Function

Annexin V, a calcium-dependent phospholipid-binding protein, can bind with very high affinity to 1PS-containing phospholipid bilayers, such as activated platelets and apoptotic cells. If annexin V was conjugated with thrombolytic agent, such as urokinase, it will lead the urokinase to the thrombus site to dissolve it. In this study, we designed and expressed a fusion protein (2UKAV) using baculovirus expression system in both attached culture and supernatant culture. At the same time, we designed two signal peptides, 3mel and 4egt, and cloned them into the 5' terminal of UKAV. They successfully secreted and enhanced the expression of UKAV. The total urokinase activity of UKAV expressed by pHT mel-UKAV and pHT egt-UKAV is at least one fold higher than that expressed by pHT UKAV. Western blot indicated that the molecular weigh of UKAV was about 68kDa. The fibrin plate assay and the membrane binding assay showed that all the UKAV display both the urokinase activity and membrane binding activity of their parent components. Our work suggested that UKAV is possible to be developed as a kind of thrombus-targeting agent and it is feasible to obtain large quantity of functional UKAV.Meanwhile, the expression vectors pT7473 UKAV and pET32 UKAV were constructed and the recombinant UKAV was expressed in E.coli HMS174(DE3). Then UKAV was purified by Ni-NTA resin. The fibrin plate assay showed that the urokinase activity of the purified UKAV was about 8100IU/mg.