The Protection Function of the PAI-2 from TNF-alpha is Dependent upon Its Protein Binding Domain.

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.

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.