Construction, expression, and characterization of a recombinant annexin B1-low molecular weight urokinase chimera in Escherichia coli.
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.
Yan
Hong-Li
HL
Department of Medical Genetics, Second Military Medical University, Shanghai 200433, China.
Wang
Wei-Ting
WT
He
Yan
Y
Zhao
Zhuan-You
ZY
Gao
Yuan-Jian
YJ
Zhang
Yi
Y
Sun
Shu-Han
SH
D000595:Amino Acid Sequence; D000818:Animals; D017302:Annexins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000627:therapeutic use*; D003328:Coronary Thrombosis / Q000188:drug therapy*; D004285:Dogs; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005343:Fibrinolytic Agents / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism / Q000627:therapeutic use; D015801:Helminth Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000627:therapeutic use*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D015202:Protein Engineering / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification / Q000627:therapeutic use; D016896:Treatment Outcome; D014568:Urokinase-Type Plasminogen Activator / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000627:therapeutic use*
D017302:Annexins; D005343:Fibrinolytic Agents; D015801:Helminth Proteins; D011993:Recombinant Fusion Proteins; C449006:annexin B1, Taenia solium; D014568:Urokinase-Type Plasminogen Activator