High-efficiency
Expression of Prolyl Endopeptidase from Aeromonas
punctata subsp. punctata in Escherichia
coli
LI Min, XU Hao, CHEN Chang-Qing
( Shanghai Research Center of Biotechnology, the Chinese Academy of
Sciences, Shanghai 200233, China )
Abstract The open
reading frame (ORF) of prolyl endopeptidase gene from Aeromonas punctata
subsp. punctata (apPEP) was amplified by PCR in three
parts. The amplified DNA segments were ligated to form the complete apPEP
gene, and then cloned into expression vectors pBL (temperature inducible) and
pKKH (IPTG inducible), respectively. After induction, the expression amounts of
recombinant apPEP in BL21/pKKH-PEP and BL21/pBL-PEP were
about 30% of the total bacterial proteins, and the enzyme activities were 100
fold higher than wild strain. Expressed apPEP was mainly soluble intracellular
protein. Non-reduced SDS-PAGE analysis showed that it was a monomer with
molecular weight about 76 kD, which corresponded to the prediction from gene
sequence. Recombinant apPEP was purified by HPLC, the purity reached 90% and
specific activity was 67 u/mg. The N-terminal analysis of apPEP demonstrated
that the protein sequence was identical as predicted from gene sequence.
Key words Prolyl endopeptidase； Escherichia coli； high-efficiency expression； purification
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