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J. Microbiol. Biotechnol. 2005; 15(2): 403-411

Published online April 28, 2005

Copyright © The Korean Society for Microbiology and Biotechnology.

Characterization of the Pediocin Operon of Pediococcus acidilactici K10 and Expression of His-Tagged Recombinant Pediocin PA-1 in Escherichia coli

Gi Seong Moon , Yu Ryang Pyun 1 and Wang June Kim 1*

Food Safety Research Division Korea Food Research Institute Seongnam Kyeonggi 463-746 Korea, 1Department of Biotechnology Yonsei University Seoul 120-749 Korea

Abstract

The relationship between plasmid (~9.5 kb) and pediocin PA-1 in P. acidilactici K10 was confirmed by plasmid curing. The pediocin operon of P. acidilactici K10 was amplified by PCR (polymerase chain reaction), and the nucleotide sequence was analyzed. The sequence of the pediocin operon of P. acidilactici K10 was similar to those of P. acidilactici strains producing pediocin PA-1/ AcH. For the expression of pediocin PA-1 in E. coli, a pQEPED (pQE-30 Xa::mature pedA) was constructed. His-tagged recombinant pediocin PA-1 (-6.5 kDa) was translated by cell-free in vitro transcription and translation using pQEPED as a DNA template. Theresult of slot blotting assay showed that transcription of recombinant pedA in E. coli M15 was induced by the addition of isopropyl-$\beta$-D-thiogalactopyranoside (IPTG) at the final concentration of 1 mM. Although the recombinant pediocin PA-1 inhibited the growth of E. coli, it was expressed in the host strain and purified by nickel-nitrilotriacetic acid (Ni-NTA) metal-affinity chromatography under denaturing condition. This is the first report for the production and one-step purification of biologically active recombinant pediocin PA-1 in E. coli.

Keywords: PediocinPA-1, pediocinoperon, Pediococcusacidilactici, expressioninE.coli