Modification,Fusion Expression And Activity Analysis Of Dermaseptin B2

Purpose:Dermaseptin B2(DRS B2)is a cationic antimicrobial peptide isolated from frog skin secretions,which not only has the advantages of broad-spectrum antimicrobial activity and low hemolytic activity,but also has less possibility to induce drug resistance.In the current situation of more and more kinds of drug-resistant strains,DRS B2 shows a good prospect for clinical application.Therefore,in this study,protein engineering technology was used to modify DRS B2 by increasing hydrophobicity and the number of positive charges to improve its biological activity.Methods and results:1.Modification of the natural peptide DRS B2Scheme 1:The C-terminal glycine(Gly22)and serine(Ser30)of DRS B2were replaced by leucine to obtain the modified peptide DRS Ba.The results of analysis proceeded by bioinformatics software‘Expasy'showed that the hydrophobicity of DRS Ba had been improved.Scheme 2:The C-terminal glycine(Gly27)of DRS B2 was replaced by lysine to obtain the modified peptide DRS Bb.The results of analysis conducted by Expasy showed that the number of positive charges of DRS Bb had been increased.Scheme 3:By integrating the strategies in scheme 1 and scheme 2,the modified peptide DRS Bc was unveiled.The analysis results of Expasy software showed that the hydrophobicity and the number of positive charges of DRS Bc had been improved.2.Construction of prokaryotic expression system of DRS B2,DRS Ba,DRS Bb and DRS BcAccording to the traditional gene recombinant technology,the recombinant p ET-32a(+)-DRS B2,p ET-32a(+)-DRS Ba,p ET-32a(+)-DRS Bb and p ET-32a(+)-DRS Bc were constructed and transformed into Escherichia coli BL21(DE3)for expression,respectively.To prove that the construction of recombinant expression vectors was successful,the positive recombinant bacteria were obtained through plate screening and were identified by PCR amplification as well as sequencing method.3.Expression of fusion proteins and optimization of conditions(1)Expression and identification of four fusion proteinsFour recombinant bacteria were induced with 0.2 mmol/L IPTG for 5 h.Then,the supernatant of bacterial lysate was extracted through centrifugation and was analyzed by SDS-PAGE electrophoresis.Fortunately,the SDS-PAGE results showed that there was an obvious band near 20 k Da in each experiment,which had a correct molecular weight consistent with theoretical value.These results indicated that His-DRS B2,His-DRS Ba,His-DRS Bb and His-DRS Bc were successfully induced and expressed.(2)Separation,purification and identification of four fusion proteinsThe fusion proteins containing Polyhistidine-tag were separated and purified by Ni²⁺affinity chromatography and were identified by SDS-PAGE electrophoresis as well as Western blot.The results showed that:(1)In SDS-PAGE results,there was a clear band near 20 k Da in each experiment,which had a correct molecular weight consistent with theoretical value.(2)The positive results of Western blot experiments demonstrated that His-DRS B2,His-DRS Ba,His-DRS Bb and His-DRS Bc had been successfully separated and purified.(3)Optimization of expression conditionsTo obtain higher protein expression levels,the control variable method was used to optimize the IPTG concentration,time,temperature and bacterial concentration in the expression systems.The optimal conditions were shown in Table 3.3.4.Comparative analysis of antibacterial activityTaking advantage of micro-broth dilution method,the detection and comparation of antibacterial activity of four fusion proteins were performed using Escherichia coli(ATCC 25922)and Staphylococcus aureus(ATTC 29213)as experimental strains and ampicillin as a positive control.The experimental results showed that:(1)The antibacterial activity of His-DRS Ba against Escherichia coli was lower than that of His-DRS B2;When the concentration of His-DRS Bb and His-DRS B2 was beyond 12.5?g/m L,the antibacterial activity of His-DRS Bb against Escherichia coli was higher than that of His-DRS B2;Compared with His-DRS B2,the antibacterial activity of DRS Bc against Escherichia coli did not have dramatic difference;(2)The antibacterial activity of His-DRS Ba against Staphylococcus aureus was lower than that of His-DRS B2;Both His-DRS Bb and His-DRS Bc had antibacterial activity against Staphylococcus aureus which were superior to that of His-DRS B2.5.Comparative analysis of hemolytic activityCompared with His-DRS B2,His-DRS Ba had an increased hemolytic activity on red blood cells.When the concentration of His-DRS Ba reached1000?g/m L,its hemolytic rate was over 20%.The hemolytic activity of His-DRS Bb was lower than that of His-DRS B2,and its hemolytic rate was less than 5%at 1000?g/m L.The hemolytic activity of His-DRS Bc was not significantly different from that of His-DRS B2.Conclusion:1.We successfully obtained His-DRS B2,His-DRS Ba,His-DRS Bb and His-DRS Bc.2.The antibacterial activity of His-DRS Bb against Escherichia coli and Staphylococcus aureus was significantly improved,while its hemolytic activity on red blood cells was reduced.These results showed that scheme 2 had certain application prospects.