Biochemical Characterization of PAI-2 and Its Mutants

To study and compare the biochemical characterization of PAI-2 and its mutants, PCR and site-directed mutagenesis methods were used to generate two PAI-2 mutants, PAI-2CD and PAI-2Q, respectively. The two mutant cDNAs were inserted into prokaryotic expression vector and expressed in a special strain of E.coli, JF1125. The expected PAI-2 mutant proteins were identified by SDS-PAGE, both covering about 14% of total bacteria proteins. The antigenicity and activity inhibiting uPA of the two mutant proteins were verified to be identical with that of wild PAI-2 by using Western blot and milk-agarose plate assay and reverse milk-agarose zymograph. The harvested recombinant bacterial cells growing in 5 L fermentor were homogenized and purified by the protocols including ammonium sulfate precipitation, Sephadex G-75 gel filtration, Q-Sepharose ion-exchange chromatography and hydrophobic interaction chromatography. The purity of the purified PAI-2CD and PAI-2Q was up to 98% and 90%, the protein yield was 18.4% and 22.1%, the specific activity was 28 640 u/mg and 14 836 u/mg, respectively. The results indicate that the biochemical characterization of PAI-2 mutants was very similar to those of the wide-type PAI-2 except that PAI-2Q can not be catalyzed by tTG.