[Gene cloning and expression of prolyl endopeptidase from Aeromonas punctata]

Prolyl endopeptidase gene was cloned from Aeromonas punctata subsp. Punctata(ST-78-3-3) using activity screening method and the 3.3 kb DNA fragment containing PEP gene was sequenced. DNA sequence from 20-2092 bp was ORF region coding 690 amino acids of prolyl endopeptidase. It was a new PEP gene through gene search. The genetic engineered E. coli BL21/pGEM-PEP overexpressing recombinant Aeromonas punctata prolyl endopeptidase(apPEP) was constructed. Cultured in YH medium, the expressed apPEP was about 30% of total cellular protein, the activity was 112 times more than that of wild strain. Expressed apPEP was mainly soluble intracellular protein, about 5% of apPEP was secreted to medium. Non-reduced SDS-PAGE analysis showed it's monomer with molecular weight about 76 kD, which corresponded with prediction by gene sequence. Recombinant apPEP was purified after tube culture, the purity reached 90% and specific activity was 67 U/mg.