Production of a hybrid protein consisting of the N-terminal fragment of urokinase and the C-terminal domain of urinary trypsin inhibitor in Escherichia coli.
We have constructed a hybrid protein (ATFHI) consisting of an N-terminal fragment from urokinase (ATF) and HI-8, which is the C-terminal domain of urinary trypsin inhibitor. The fusion genes for the hybrid proteins were engineered by PCR and cloned into expression plasmids. Under the control of the tac promoter, fusion genes were efficiently expressed in Escherichia coli. The hybrid proteins, produced as inclusion bodies in E. coli, were refolded by a dialysis method and purified by ion-exchange chromatography. ATFHI exhibited bifunctional activity related to antimetastatic effects: the urokinase receptor-binding activity of ATF and the inhibitory activity of HI-8 on plasmin.
Sugino
D
D
Nissin Central Research Institute, Shiga, Japan.
Okushima
M
M
Kobayashi
H
H
Terao
T
T
D000818:Animals; D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics*; D005341:Fibrinolysin / Q000187:drug effects; D016650:Fluorescein-5-isothiocyanate; D006023:Glycoproteins / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics* / Q000378:metabolism* / Q000494:pharmacology; D014407:Tumor Cells, Cultured; D014568:Urokinase-Type Plasminogen Activator / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology
D006023:Glycoproteins; D011994:Recombinant Proteins; D005341:Fibrinolysin; D014568:Urokinase-Type Plasminogen Activator; D016650:Fluorescein-5-isothiocyanate; C028665:urinastatin