High-efficiency Expression of Prolyl Endopeptidase from Aeromonas punctata subsp. Punctata in Escherichia coli.

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.