Function Study And Quality Control Of Fusion Protein With Both Fibrinolytic And Anticoagulant Activities

1:Fermentation, purification and function study of staphylokinase-hirudin fusion protein, STH, which linked by thrombin recognition peptideObjective: To establish the high-density fermentation and purification process of staphylokinase-hirudin fusion protein, STH, which linked by thrombin recognition peptide, and to study the function of STH in vitro and in vivo. Methods: STH was expressed in Escherichia coli BL21 under a high-density fermentation. After fermentation, the E.coli bodies were harvested by centrifugation. STH was extracted from the cell pellet by six freeze-thaw cycles in 20 mM Tris-Hcl buffer (pH 8.0). The extraction was centrifuged and the supernatant was collected and purified by ultrafiltration and two-step ion-exchange chromatography. The purity of the purified production was determined by SDS-PAGE and RP-HPLC. The cleavage of STH by thrombin was monitored by SDS-PAGE and Western Blot. Antithrombin activity of STH was analyzed by amidolytic assay and fibrin clot lysis assay. Fibrinolytic activity of STH was assessed by agar-fibrin plate assay, amidolytic assay and fibrin clot lysis assay. The affinity of STH toward thrombin was estimated by thrombin-binding assay and fibrin clot lysis assay. The fibrinolytic and anticoagulant activities of STH in whole blood were analyzed by thromboelastography. And then, the thrombolytic effect and bleeding risk of STH in vivo were evaluated in rat inferior vena cava thrombosis model and mouse tail-transection model. Results: STH expression of 1.48 g/L fermentation broth was achieved after 20 hours cultivation under the high-density fermentation. In the purification technique, the activity recovery rate was 56%. After purification, the purity of the product, determined by RP-HPLC, was over 98%, and the specific activity of STH were 2.6×104 IU/mg. The results of SDS-PAGE and Western blot indicated that the linker peptide between SAK and HV2 could be successfully and specifically recognized and cleaved by thrombin. The results of amidolytic assay and fibrin clot lysis assay showed that the N-terminus of HV2 in STH was blocked by staphylokinase and linker peptide, impeding HV2's anticoagulant activity. Once cleaved, STH displayed 35.68% of the anticoagulant activity of equimolar HV2 and exhibited anticoagulant effect in fibrin clot lysis assay. Amidolytic assay and thromboelastogram assay showed that the fibrinolytic activity of STH was comparable to SAK. Thrombin-binding and fibrin clot lysis assays showed that the C-terminus of HV2 retained its high affinity for thrombin. Moreover, compare with SAK, STH showed improved thrombolytic effects and a lower bleeding risk in animals. Conclusion: A simple, high efficiency of STH production process is established, and the process is easy to scale up. The results in vitro and in vivo study indicate that STH is a promising thrombolytic agent that may have the capacity to display bifunction and thrombus-targeted release of anticoagulant activity in vivo, and thereby reduce the risk of systemic bleeding.2: Quality control of staphylokinase-hirudin fusion protein, SFH, which linked by FXa recognition peptideObjective: To establish the quality control methods of staphylokinase-hirudin fusion protein, SFH, which linked by FXa recognition peptide. Methods: According to the third pharmacopoeias of the People′s Republic of China (2005) and the relevant directions about biological products, the detection methods of the following items of SFH stock solution were established: the protein concentration was monitored by Lowry method; the purity was detected with non-reducing SDS-PAGE and RP-HPLC; antibiotic residues was monitored by lysozyme circle; anticoagulant activity was analyzed by amidolytic assay. Results: The protein concentration of three batches of SFH stock solution was determined by Lowry methods, and the results were 18.48, 24.85 and 23.96 mg/ml, respectively, which all in line with the standard of more than 15mg/ml. Non-reducing SDS-PAGE assay, three batches of SFH stock solution putity results were 96%, 97% and 97%, respectively, which all in line with the standard of more than 95%. RP-HPLC assay results of three batches of SFH stock solution were 99.32%, 100% and 100%, respectively, which all in line with the standard of more than 95%. The antibiotic residues of three batches of SFH stock solution were determined by lysozyme circle, and results were negative. Amidolytic assay, the anticoagulant activities of three batches of SFH stock solution which were cleaved by FXa at 0h were 3.32%, -7.63% and 3.77%, respectively, which all in line with the standard of±10%. Amidolytic assay, the anticoagulant activities of three batches of SFH stock solution which were cleaved by FXa at 3h were 72.24%, 60.72% and 65.08%, respectively, which all in line with the standard of more than 50%. Conclusion: The methods of determination of Protein concentration, purity, antibiotic residues and anticoagulant activity of SFH stock solution are established. And according to the quality control methods, the quality of three batch of SFH stock solution are examined and the results show that the quality of STH stock solution are consistent with the regulations of the National institute for the control of pharmaceutical and biological products about biological product.