HETEROLOGOUS EXPRESSION AND PURIFICATION OF NisA, THE PRECURSOR PEPTIDE OF LANTIBIOTIC Nisin FROM LACTOCOCCUS LACTIS


Karakas-Sen A. , Narbad A.

ACTA BIOLOGICA HUNGARICA, vol.63, no.2, pp.301-310, 2012 (Journal Indexed in SCI) identifier identifier identifier

  • Publication Type: Article / Article
  • Volume: 63 Issue: 2
  • Publication Date: 2012
  • Doi Number: 10.1556/abiol.63.2012.2.11
  • Title of Journal : ACTA BIOLOGICA HUNGARICA
  • Page Numbers: pp.301-310

Abstract

The lantibiotic nisin is a ribosomally synthesised and post-translationally modified antimicrobial peptide produced by strains of Lactococcus lactis, and used as safe and natural preservative in food industry. The nisA structural gene encodes ribosomally synthesised and biologically inactive a 57 amino acid precursor peptide (NisA) which undergoes several post-translational modifications. In this study, we report the expression of precursor nisin as a His6-tagged peptide in Escherichia coli and its purification using a nickel affinity column. The technique of spliced-overlap extension PCR was used to amplify the nisA gene and the T7 promoter region of pET-15b vector. This approach was used to introduce six histidine residues at the C-terminus of prenisin. The identity of the expressed peptide was confirmed by N-terminal sequencing. The expressed His-tagged prenisin was purified under denaturing conditions, and named as prenisin-His6. The purified prenisin-His6 was analyzed by SDS-PAGE, Western blotting and mass spectroscopy. These results showed that the nisin precursor peptide can be successfully produced using an E. coli expression system.