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To produce a thrombi-targeting plasminogen activator, low molecular weight single-chain urokinase gene (scuPA32k) was spliced with the full-length cDNA of annexin B1 gene (anxB1) by overlap extension method. The fused gene anxB1scuPA was ligated into pET28a vector, transformed into E. coli BL21-RIL, and then induced to express under the control of T7 promoter. The AnxB1ScuPA protein ex- pressed amounted to 22% of the total bacterial proteins. The product was refolded, and then purified by using DEAE Sepharose fast flow ion-exchange column and Superdex S-200 gel-filtration column. HPLC analysis revealed that the final purity is about 95%. The specific activity of AnxB1ScuPA, measured as amidolytic activity, reached 100,000 IU/mg. It had a similar S2444 catalytic efficiency (kcat/Km) to ScuPA32k, and also showed high activated-platelet membrane-binding activity and anticoagulant activity, indicating that the chi- mera fully retained the components of enzymatic and membrane-binding activities of the parent molecules. In vivo test revealed that, the dogs administered with AnxB1ScuPA had less reperfusion time, higher reperfu- sion ratio, and less bleeding effects than those with urokinase. These findings indicated that AnxB1ScuPA might have advantages over current available thrombolytic agents.
The fused gene anxB1scuPA was ligated into pET28a vector. The fusion gene anxB1scuPA was ligated into pET28a vector, and the full-length cDNA of annexin B1 gene (anxB1) was amplified by overlap extension method. To produce a thrombi-targeting plasminogen activator, low molecular weight single-chain urokinase gene (scuPA32k) was spliced with the full- transformed into E. coli BL21-RIL, and then induced to express under the control of T7 promoter. The AnxB1ScuPA protein ex- pressed amounted to 22% of the total bacterial proteins. The product was refolded, and then purified by using DEAE Sepharose fast The specific activity of AnxB1ScuPA, measured as amidolytic activity, reached 100,000 IU / mg. It had a similar S2444 catalytic activity efficiency (kcat / Km) to ScuPA32k, and also showed high activated-platelet membrane-binding activity and anticoagulant activity, indicating that the chi- mera fully retained the components of e In vivo test revealed that the dogs administered with AnxB1ScuPA had less reperfusion time, higher reperfu- sion ratio, and less bleeding effects than those with urokinase. These findings indicate that AnxB1ScuPA might have advantages over current available thrombolytic agents.