Heterologous expression and purification of NisA, the precursor peptide of lantibiotic nisin from Lactococcus lactis.
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.
Institute of Food Research, Norwich Research Park, Colney, Norwich NR4 7UA, UK. akarakas@uludag.edu.tr
Asuman
A
Karakas-Sen
A
A
Narbad
D001430:Bacteriocins / Q000378:metabolism*; D002846:Chromatography, Affinity; D004926:Escherichia coli / Q000378:metabolism; D005822:Genetic Vectors; D013294:Lactococcus lactis / Q000235:genetics*; D009561:Nisin / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis
D001430:Bacteriocins; D011993:Recombinant Fusion Proteins; D009561:Nisin; C102526:nisin A