Heterologous expression and purification of nisa, the precursor peptide of lantibiotic nisin from lactococcus lactis
Date
2012-06
Authors
Narbad, Arjan
Journal Title
Journal ISSN
Volume Title
Publisher
Akademiai Kiado Zrt
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.
Description
Keywords
Life sciences & biomedicine - other topics, Nisin, Prenisin, His-tag, Purification, Spliced overlap extension, Posttranslational modification, Escherichia-coli, Biosynthesis, Gene, Dehydration, Proteins, Cloning
Citation
Karakaş Ş. A. ve Narbad, A. (2012). "Heterologous expression and purification of nisa, the precursor peptide of lantibiotic nisin from lactococcus lactis". Acta Biologica Hungarica, 63(2), 301-310.