Biochemical
Characterization of PAI-2 and Its Mutants
TIAN Yu, SHEN Jun-Qing, LI Ping, ZHU Yun-Song
( Laboratory of Molecular Genetics, Basic Medical School, Shanghai Medical
University, Shanghai 200032, China )
Abstract 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.
Key words plasminogen activator inhibitor
type-2；
mutation；
expression and purification
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