Expression Of Modified Dermaseptin B2 In Escherichia Coli And The Detection Of Its Antibacterial Activity

Objective:the natural antimicrobial peptide Dermaseptin B2 not only processes broad antibacterial activity,but also processes the advantages of anti-tumor activity and low hemolytic activity.Meanwhile,it has no toxicity to some animal cells.So it has a good development prospect.Previous studies have shown that the antibacterial activity of Dermaseptin B2 depends on the number and side chain size of hydrophobic residues on the non-polar helix surface of N-terminal and C-terminal.In this study,Dermaseptin B2 was modified by replacing serine at N-terminal and C-terminal with leucine.After bioinformatics analysis,the prokaryotic expression system of Dermaseptin B2 and the modified peptide Dermaseptin B2~m was constructed,and preliminary comparative study on the antimicrobial activity was conducted.Methods and results:1.Modification of natural antimicrobial peptides Dermaseptin B2Previous studies have shown that Dermaseptin B2 can improve its antibacterial activity by increasing the number of hydrophobic residues at the N-terminal and C-terminal,or by replacing the hydrophobic residues of small side chains with large side chains.In this study,the modification scheme was as follows:first,the serine of Dermaseptin B2 N-terminal and C-terminal was replaced by leucine,and the bioinformatics analysis showed that the hydrophobicity of Dermaseptin B2 was improved.Second,mRNA sequence of Dermaseptin B2obtained from NCBI Gene bank was converted into cDNA sequence,and intestinal kinase cleavage site(5'-GACGACGACGACAAG-3')was added at its 5'end for the convenience of subsequent protein isolation and purification.2.Construction of Dermaseptin B2 and Dermaseptin B2~m prokaryotic expression system(1)Construction of fusion protein expression vectorDermaseptin B2 and Dermaseptin B2~m prokaryotic fusion expression vector were constructed according to the classical gene cloning technique by using pET-32a(+)as the expression vector,Escherichia coli BL21(DE3)as the host strain,and isopropylthiogalactoside(IPTG)as the inducer.The results showed that the fusion protein expression vector was successfully constructed identified by resistance screening and sequencing.(2)Expression and identification of fusion proteinExpression and identification:After we successfully transferred the recombinant vector into competent Escherichia coli BL21(DE3),final concentration of 0.5 mmol/L IPTG was added to induce expression for 5 h at 37?.The protein was extracted and identified by SDS-PAGE,with the protein of Escherichia coli BL21(DE3)as control.The obvious band near 20 KDa can be observed,which was consistent with the theoretical molecular mass,that is,the target fusion protein was obtained.Isolation and purification:After the recombinant bacteria was induced by IPTG,the supernatant and precipitate portions of the bacterial were extracted respectively.The results showed that the soluble components of Dermaseptin B2 and Dermaseptin B2~m were more than50%by SDS-PAGE,indicating that the fusion protein was mainly distributed in the bacterial cell fluid.The fusion protein was separated and purified by affinity chromatography after the bacteria was lysed and the supernatant was obtained by centrifugation.SDS-PAGE analysis showed that there was a single obvious band near 20 KDa,which was consistent with the theoretical molecular mass.Optimization of expression system:We analyzed several factors affecting the expression of fusion protein,including the concentration of IPTG(0.1-2 mM),the induction time(3-12 h),the induction temperature(25?,30?and 37?).By this method,we obtained optimal expression condition of Dermaseptin B2 with the final concentration of 0.8 mM IPTG,incubated for 5 h at25?and optimal expression condition of Dermaseptin B2~m with the final concentration of 0.5mM IPTG,incubated for 7h at 25?.3.Separation and purification of target peptideThe isolated and purified fusion protein was digested with enterokinase in a Ni-chelating column at 25?for 9 h,and the flow-through solution(that is,the target peptide solution)was collected.Tricine-SDS-PAGE showed that the protein is a molecular weight of about 5 KDa,indicating that the affinity-purified target peptide was obtained.4.Comparative study on antibacterial activityThe antibacterial activities were detected by micro-broth dilution methods by using Escherichia coli(DH5?)and Staphylococcus aureus(ATTC 29213)as experimental strains,and ampicillin as a positive control.The results showed that:(1)Dermaseptin B2~m had higher antibacterial activity against Escherichia coli than Dermaseptin B2 and ampicillin at a concentration greater than 2.5?g/mL.Dermaseptin B2 had higher antibacterial activity against Escherichia coli than ampicillin;(2)Dermaseptin B2~m has antibacterial activity against Staphylococcus aureus,but its antibacterial activity is lower than that of Dermaseptin B2 and ampicillin.These results suggests that Dermaseptin B2~m has highly effective against Gram-negative bacteria.Conclusion:In this study,we successfully constructed the prokaryotic expression system of Dermaseptin B2 and Dermaseptin B2~m,and acquired the antimicrobial peptides Dermaseptin B2 and Dermaseptin B2~m.When the concentration was higher than 2.5?g/mL,Dermaseptin B2~m was more effective than Dermaseptin B2 and ampicillin against E.coli(DH5?).Dermaseptin B2 was more effective than ampicillin against E.coli(DH5?).What's more,Dermaseptin B2~m has antibacterial activity against S.aureus(ATCC 29213),but its antibacterial activity was lower than Dermaseptin B2 and ampicillin.Dermaseptin B2~m has highly antibacterial activity against Gram-negative bacteria.