Soluble expression of a strong thrombolytic pro-urokinase mutant in Escherichia coli.
A recombinant pro-urokinase mutant GZ5-sPA was successfully constructed by fusion of a high fibrin-affinity fragment GZ5 to the N-terminus of the serine protease domain of pro-urokinase (sPA). The fragment GZ5 contains a tetrapeptide GPRP and a tripeptide RGD, and was synthesized in bacterial preferred expressing codons. The mutant was then fused to the C-terminus of maltose binding protein (MBP) carried by pMAL-C2x vector, and expressed in Escherichia coli strain Origami (DE3). The produced fusion protein was highly soluble in the cytoplasm of the bacteria. After being cleaved with PreScission Protease to remove MBP tag, GZ5-sPA showed a molecular weight of 31 kDa on SDS-PAGE. GZ5-sPA maintained the same epitope as wild-type pro-urokinase and possessed a thrombolytic activity three times higher than standard urokinase did after being activated as two-chain form. The results could be a clue to other complicated heterogenous proteins similar to pro-urokinase.
Lin
Jian
J
College of Life Sciences, Biopharmaceutical Centre, Sun Yat-Sen University, Guangzhou 510275, China.
Yang
Xiaoyi
X
Deng
Riqiang
R
Yu
Boguang
B
Lai
Huangjie
H
Sun
Weili
W
Wu
Wenyan
W
D015153:Blotting, Western; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D005337:Fibrin / Q000378:metabolism; D005343:Fibrinolytic Agents / Q000378:metabolism*; D005822:Genetic Vectors / Q000378:metabolism; D007700:Kinetics; D058619:Maltose-Binding Proteins; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009154:Mutation; D010974:Platelet Aggregation; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012995:Solubility; D014568:Urokinase-Type Plasminogen Activator / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism
D002352:Carrier Proteins; D005343:Fibrinolytic Agents; D058619:Maltose-Binding Proteins; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D005337:Fibrin; D014568:Urokinase-Type Plasminogen Activator; C067551:saruplase