Purification And Functional Analysis Of Nisin Resistance Protein (NSR) Mutation Expressed In Escherichia Coli

Nisin is a lantibiotic antimicrobial peptide produced by some strains of Lactococcus lactis. It is highly active against a wide range of Gram-positive bacteria including important food pathogens and relevant clinical bacteria. in some nisin non-producing L. lactis strains, nisin resistance could be conferred by a specific nisin resistance gene (nsr) which encodes a 35 kDa nisin resistance protein (NSR).Preliminary studies in our laboratory showed that as the first identified tail-specific protease in L. lactis, NSR endows host strains with resistance to nisin through degrading the bactericidal agent to less potent products, which cleave nisin in vitro and the cleavage site was pinpointed between methyllanthionine (MeLan)28 and Ser29 .On the basis of this study, this article further on reveal the relationship between the structure and the function of NSR, and to further understand the nisin resistance mechanism conferred by NSR. The current study paves the way for in-depth functional studies on NSR.First of all, this study is to determine the NSR core function of in vitro degradation Nisin. On the basis of the homologous protein sequence analysis and secondary structure prediction, tertiary structure blast, construction NSR different domain of GST-fusion expression vector; NSR-TSP,NSR△94,NSR△78,NSR△67,NSR△38 was overexpressed in Escherichia coli by fusion with GST, purified truncation were obtained through glutathione (GSH) affinity chromatography followed by cleavage of GST tag. Analysis their nisin degradation functions in vitro. Antimicrobial activity analysis revealed that nisin lost its bactericidal activity after incubation with NSRΔ38. Further reversed-phase high performance liquid chromatography (RP-HPLC) analysis indicated that NSRΔ38 displayed proteolytic activity against nisin, thus inactivating the antimicrobial peptide. The results showed that NSRΔ38 have Nisin activity caused by the degradation, and truncation NSR-TSP, NSR△94, NSR△78, NSR△67 does not have the activity of Nisin degradation.For further analysis to identify different functional areas combined with Nisin, using the same strategy to construct the different truncation of site-directed mutagenesis of Ser236 in NSR, NSRS△94, NSRS△78, NSRS△67, NSRS△38. Overexpressed in Escherichia coli by fusion with GST, purified truncation were obtained through glutathione (GSH) affinity chromatography followed by cleavage of GST tag to obtain the pure non-degradable mutant protein. Use of surface plasmon resonance (SPR) to detect NSRS△27, NSRS△38, NSRS△94 mutants the binding capacity of Nisin in vitro. The results showed that the Kd of NSRS△38 and NSRS△94 mutant are similar, while the Kd of NSRS△27 is higher than NSRS△38 and NSRS△94 an order of magnitude, indicating that the stronger binding capacity of the substrate Nisin.