Expression of a fibrinolytically active human pro-urokinase fusion protein in Escherichia coli.
The gene encoding human pro-urokinase(pro-UK) was cloned into plasmid pEZZ318 and fused to the gene coding for the signal peptide of staphylococcal protein A and IgG bindinging domain. The fusion protein which was synthesized under the control of T7 promoter in Escherichia coli and secreted into the growth medium, was found to be fibrinolytically active. Approximately 60% of the total activity was secreted into the culture medium, where levels of activity approached 150,000 I.U./liter and about 40% of the total activity remained in the cell lysate with levels of activity around 100,000 I.U./liter. The fusion protein was purified in a single step by IgG affinity chromatography. These results demonstrate that human pro-UK can be synthesized and secreted by E. coli as a fibrinolytically active fusion protein.
Hua
Z
Z
Department of Biochemistry, Nanjing University, People's Republic of China.
Jie
L
L
Zhu
D
D
D001665:Binding Sites; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004792:Enzyme Precursors / Q000096:biosynthesis; D004926:Escherichia coli; D005342:Fibrinolysis; D006801:Humans; D007074:Immunoglobulin G; D008970:Molecular Weight; D010957:Plasmids; D021382:Protein Sorting Signals / Q000096:biosynthesis; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D013205:Staphylococcal Protein A / Q000096:biosynthesis / Q000302:isolation & purification; D014568:Urokinase-Type Plasminogen Activator / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism*
D004792:Enzyme Precursors; D007074:Immunoglobulin G; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D013205:Staphylococcal Protein A; D014568:Urokinase-Type Plasminogen Activator; C067551:saruplase