To express the fusion protein ATF-PAI2CD (urokinase-type plasminogen activator amino terminal fragment-plasminogen activator inhibitor type 2 with the region inter C and D helices deleted) gene in E. coli and determine the biological characterization of fusion protein ATF-PAI2CD, the cDNA fragment encoding ATF-PAI2CD was cloned into the expression vector pLY-4 and transformed into E. coli JF1125. After temperature induction, the expression amount of ATF-PAI2CD account for 15% of total bacterial protein. The result was confirmed by Western blot. ATF-PAI2CD protein was isolated and purified by washing and solubilization of inclusion body, renaturation and ion exchange chromatography. The final product displayed a single band with a corresponding molecular weight 62 kD in SDS-PAGE. The purity was over 90%, the protein yield was 50% and the specific activity was 12 000 IU/mg. The PAI activity was measured by chromogenic assay. The purified fusion protein inhibited urokinase-type plasminogen activator as measured by milk-agarose plate assay, and bound to human lung cancer cells via uPA receptor (uPAR), which was confirmed by radio competition experiments. The results indicate that the biological characteristics of ATF-PAI2CD were very similar to those of the wide type PAI-2 (or mutants PAI-2, PAI-2CD) and to pro-uPA in binding to uPAR-bearing cells.
|Original language||English (US)|
|Number of pages||5|
|Journal||Acta Biochimica et Biophysica Sinica|
|State||Published - 2003|
- Fusion protein
- Radio ligand receptor binding assay
ASJC Scopus subject areas