(03374)Construction,Expression, and Characterization of a Recombinant Annexin B1-Low Molecular Weight Urokinase Chimera in Escherichia coli

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 (k_cat/K_m)
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

—————–
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, [email protected]