[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.
Li
M
M
Shanghai Research Center of Biotechnology, Chinese Academy of Sciences, Shanghai 200233.
Chen
C
C
Wang
D
D
D000333:Aeromonas / Q000201:enzymology* / Q000235:genetics; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D008969:Molecular Sequence Data; D000085822:Prolyl Oligopeptidases; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000378:metabolism; D012697:Serine Endopeptidases / Q000096:biosynthesis* / Q000235:genetics* / Q000378:metabolism
D011994:Recombinant Proteins; D012697:Serine Endopeptidases; D000085822:Prolyl Oligopeptidases