Fermentation And Purification Of Genetic Engineering Strains Producing Lysozyme C-terminal Peptide From Stichopus Japonicus

Like most subsistent lysozymes of c-type and g-type in nature,the lysozyme of sea cucumber Stichopus japonicus(SjLys)can also hydrolyse the ?-1,4 glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine of the peptidoglycan which is an important constituent of bacterial cell wall.As a result the bacteria will be dead when it lacks the protection of cell wall.This project had demonstrated that the antimicrobial activity of SjLys would increase when it was incubated in the condition of high temperature,but its molecular mechanism was still unknow.This study used the C-terminal domains of the lysozyme from the sea cucumber S.japonicus(SjLys-C)to discuss the effect of temperature variation on the antibacterial activity.Firstly,in this research,we constructed a new recombinant plasmid pET-30a(+)-SjLys-C and expressed successfully in E.coli Rosetta(DE3)pLysS.In addition,the research also optimizes the old constructed recombinant plasmid pET-32(+)-SjLys-C expression system.We transformed the recombinant plasmid pET-32(+)-SjLys-C into a new expression host E.coli Rosetta-GamiB(DE3)pLysS which can help the correct folding of disulfide in the protein,and the recombinant protein SjLys-C can be expressed highly in vitro.In the new expression host,induced by IPTG,majority of the recombinant protein can be expressed in soluble form,and has the antibacterial activity.Finally,we get a 92%purity protein using Ni2+ affinity chromatography and Sephadex G-25 dextran gel chromatography.Based on the antibacterial test,the fusion protein SjLys-C produced by new host displayed an inhibitive effect on the growth of four tested bacteria.In addition,the antimicrobial activities on three tested bacteria were enhanced about 5%-22%after the fusion protein was heated in 100? for 40 minutes.Finally,this study has used molecular dynamics simulation(MD)in order to simulate the changes in the internal structure of the protein to understand the changes of the SjLys-C protein structure.We compared the simulated changes of root mean square deviation(RMSD),root mean square fluctuation(RMSF),radius of gyration(Rg)and protein tertiary structure at the conditions of 30? and 100?.It was found that the RMSD had small changes,while four areas of RMSF fluctuations and the value of Rg in 100? was slightly less than that in 30?.According to the simulation results,the overall tertiary structure of the protein after heat treatment did not change too much,only a part of the structure near a small part of protein sequence of N-terminus was adjusted.It was also found that the value of the distance between two active sites of 100? was less than that of 30?.By low temperature annealing simulation,it can be found that the protein structure remained stable after annealing except a small region near the N-terminal of the protein which only have tiny changes.The results might discover a new interpretation of the antibacterial function to the fusion protein SjLys-C which was treated on high temperature.