RESUMO
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.
RESUMO
The cDNA of PAI-2 mutants, PAI-2CD and PAI-2Q were inserted into eukaryotic expression vector, pcDNA3, producing pZLE-PAI2CD and pZLE-PAI2Q respectively. The PAI-2 mutant expression plasmids were transfected into HeLa cells and the transfected cells expressing mutant proteins were selected by G418, Northern blot and ELISA assay. The ELISA assay showed that the expression level of PAI-2 mutant protein was equal to that of wild type PAI-2 in transfected cells. The biochemical characteristics of PAI-2 mutant proteins were very similar to that of the wild type PAI-2. The results of crystal violet and MTT assays showed that the PAI-2 mutants did not protect cells from the cytotoxic effects of TNF-alpha. The results of this study indicate that the protection of PAI-2 from apoptosis induced by TNF-alpha is dependent upon the protein-protein interaction mediated by the CD-interhelical domain of PAI-2. The tTG-mediated interaction between PAI-2 and cytosol protein might play a leading role in protecting from apoptosis induced by TNF-alpha.
