[Biological function of fusion protein ATF-PAI2CD].
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.
Wang
Xia
X
Department of Molecular Genetics, Shanghai Medical College, Fudan University, Shanghai 200032, China.
Li
Ping
P
Zhang
Yu-Qing
YQ
Hou
Min
M
Sun
Xing-Hui
XH
Tan
Li
L
Zhu
Yun-Song
YS
D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D008175:Lung Neoplasms / Q000378:metabolism / Q000473:pathology; D010446:Peptide Fragments / Q000378:metabolism*; D017396:Plasminogen Activator Inhibitor 2 / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism*; D014568:Urokinase-Type Plasminogen Activator / Q000378:metabolism*
D010446:Peptide Fragments; D017396:Plasminogen Activator Inhibitor 2; D011993:Recombinant Fusion Proteins; D014568:Urokinase-Type Plasminogen Activator