Heterologous Expression Optimization And Antibacterial Effect Of Listeria Monocytogenes P60 Protein

Listeria monocytogenes(Lm)is a zoonotic pathogen that is widely found in nature.It is also a foodborne pathogen that can cause listeriosis in humans and animals.The presence of Listeria monocytogenes in food threatens human safety.It can grow and thrive in low-temperature environments and is one of the major pathogens that threaten human health.The Lm-p60 protein is an extracellular protein produced by Listeria monocytogenes.It is encoded by the iap gene and is closely related to the pathogenicity of Listeria monocytogenes.The Lm-p60 protein has the activity of amidase,which can hydrolyze the bacterial cell wall,and has a high research value.The tertiary structure prediction of Lm-p60 protein and the study of its domain function showed that it possesses two independent structural domains,N-terminal domain and C-terminal domain.The N-terminal domainis comprised of two LysM motifs and one SH3 motif.The C-terminal contains only one N1pC/P60 family domain.In the process of lysing the bacterial cell wall,only LysM motif and SH3 motif was involved in substrate recognition and binding,thereby confirming the function of the N-terminal domain in recognizing and binding the substrate,while the C-terminal domain was identified as catalytic domain.At present,it is not clear whether Lm-p60 protein has antibacterial activity,and Lm-p60 protein still has problems of insufficient expression and low activity in the process of heterologous expression.In order to obtain a large number of high-activity Lm-p60 proteins,in this study,SDS-PAGE analysis was used to compare the recombinant protein expression of different vectors to determine the optimal expression vector,and the results showed that the optimal expression vector was for Lm-p60 protein pET30a.Subsequently,this study identified the optimal heterologous expression conditions for Lm-p60 protein.The results showed that the optimal expression process determined was that the host strain was enriched to a OD600nm value at 0.553 and IPTG was then added to a final concentration of 0.5 mmol/L followed by 4h expression on a shake table with a shaking speed of 160 rpm at 30 ?.The optimum time for ultrasonic breakage is 20 min.Lm-p60 protein was purified under the best condition by nickel column affinity chromatography.The purification rate of Lm-p60 protein was 1.2,and the recovery rate was 95%.Bacterial drug resistance problem has become increasingly prominent and the development of new antibacterial agents is urgent.Lm-p60 protein has the activity of lysing bacterial cell walls.It has become an important work to study whether Lm-p60 protein has antibacterial and antibacterial effects.In this study,Micrococcus sclerotiorum was used as indicator bacteria.The agar-perforated method confirmed that Lm-p60 protein had antibacterial activity,and its antibacterial study on substrate binding properties of Lm-p60 protein become another crucial aspect of our work.Lysozyme,as a relatively thorough research antibacterial enzyme,has principle of cell wall lysis from that of Lm-p60 protein.Based on this study,it can be concluded that the combined use of Lm-p60 protein with with lysozyme could enhance the antibacterial effect of lysozyme,providing good materials for lysozyme modification.In this study,the N-terminal domain and LysM2-C domain of Lm-p60 protein was also constructed with pET30a vector,and the N-terminal domain was also found to have cleavage activity by turbidimetric assay,but the antibacterial effect was not obvious.However,the cleavage activity of p60LysM2-Cter recombinant protein is very weak.