Cloning and Nucleotide
Sequencing of Prolyl Endopeptidase Gene
from Aeromonas punctata subsp. punctata*
SHEN Guo-Xiang and SHI Ji-Ping
( Department of Biosynthetic Pharmceutics, School of Pharmacy, Shanghai
Medical University, Shanghai 200032, China )
Abstract Prolyl endopeptidase activity
was found in Aeromonas punctata subsp. punctata. The genomic DNA was
partially digested with EcoRI and the recovered 8～16 kb DNA fragments were
inserted into the EcoRI site of plasmid pUC19, and were transformated
into Escherichia coli DH5α. The resulted clones were screened by using
Benzyloxycarbonyl-Gly-Pro-β-naphthylamide, the specific substrate of prolyl
endopeptidase and a positive clone was obtained. The 12 kb insertion fragment
of recombinant plasmid was digested with HincII and subcloned. The PEP
gene was found in the 3.5 kb HincII/EcoRI fragment.
Nucleotide sequence of the gene was completely sequenced by Auto Sequencer. The
complete gene consisted of 2 073 bp corresponding to 690 amino acid residues
with a calculated molecular weight of 76 467 Da. The amino acid sequence was
92.3%、53.2%、33.5%、33.2% and 20.5% homologous to
those of Aeromonas hydrophila, Flavobacterium meningosepticum,
porcine brain, human lymphocytes and Pyrococcus furiosus respectively.
From a survey of sequence homology with other members of the prolyl
endopeptidase family, the amino acid residues involved in the catalytic triad
were deduced to be Ser538、Asp622 and His657.
Key Words prolyl endopeptidase； serine proteinases； cloning-molecular； nucleotide sequence
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