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ISSN 0582-9879 Acta Biochim et Biophysica Sinica 2004, 36(3):184-190 CN 31-1300/Q
Hong-Li YAN, Wei-Ting
WANG1, Yan HE , Zhuan-You ZHAO1, Yuan-Jian GAO, Yi ZHANG, and Shu-Han SUN*
(Department of Medical Genetics, Second Military Medical University, Shanghai
200433, China;
1Department of Pharmacology, Tianjin Institute of Medicine, Tianjin 300193,
China)
Abstract 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 expressed 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 chimera 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 reperfusion ratio, and less bleeding effects than those with urokinase. These findings indicated that AnxB1ScuPA might have advantages over current available thrombolytic agents.
Key words annexin B1; thrombolytic agent; low molecular weight single-chain urokinase; chimera
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Received: November 7, 2003 Accepted:
January 13, 2004
This work was supported by the grants from the National High Techno-logy Research
and Development Program of China (No. 101-06-05-04) and the National Natural
Science Foundation of China (No. 30271167)
*Corresponding author: Tel, 86-21-25070332; Fax, 86-21-25070331; E-mail, shsun@smmu.edu.cn