Microbial Biotechnology

Electronic Journal of Biotechnology ISSN: 0717-3458 Vol. 10 No. 4, Issue of October 15, 2007
© 2007 by Pontificia Universidad Católica de Valparaíso -- Chile Received January 3, 2007 / Accepted May 24, 2007
DOI: 10.2225/vol10-issue4-fulltext-11

Heterologous expression, purification and refolding of an anti-listerial peptide produced by Pediococcus acidilactici K7

Prakash M. Halami*
Department of Food Microbiology
Central Food Technological Research Institute
Mysore 570 020 India
Tel: 91 821 2517 539
Fax: 91 821 2517 233
E-mail: foodmicro@cftri.res.in

Arun Chandrashekar
Department of Plant Cell Biotechnology
Central Food Technological Research Institute
Mysore 570 020 India
Tel: 91 821 2516 501
Fax: 91 821 2517 233
E-mail: pcbt@cftri.res.in

Website: http://www.cftri.com

*Corresponding author

Financial support: This work was supported by the financial assistance obtained from the institute funds of CFTRI, Council of Scientific and Industrial Research, New Delhi. Government of India.

Keywords: fusion protein, inclusion bodies, in vitro refolding, pediocin PA-1, Pediococcus acidilactici, RP-HPLC.


AU: arbitrary unit
BHI: brain heart infusion
β-ME: β-mercaptoethanol
DTT: dithiotritol
IBs: inclusion bodies
LAB: lactic acid bacteria
LB: Luria Bertani
MIC: Minimum inhibitory concentration
MRS: de Man, Rogosa and Sharpe
MW: molecular weight
Ni-NTA: Nickel- nitrilotriacetic acid
PCR: polymerase chain reaction

Full Text

The fusion protein, 6XHis-Xpress-PedA was constructed and expressed in Escherichia coli BL21 (DE3). The presence of a 12.8 kDa recombinant protein, localized in inclusion bodies (IBs) at high concentration, was confirmed by SDS-PAGE analysis and by western blotting using anti-His antibody. The rec-pediocin was purified by Nickel-nitrilotriacetic acid beads and refolded using 5 mM of β-mercaptoethanol along with 1 M glycine. Results indicated that the refolded rec-pediocin had an early elution profile in the RP-HPLC when compared to the unfolded protein and it exhibited biological activity against Listeria monocytogenes V7 which was approximately 25 times less active compared to native counterpart. The final yield of purified rec-pediocin was 3 mg/l of the culture and is estimated to be 8-10 times higher than the purification by conventional methods.

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