Expression,purification And Activity Of Antimicrobial Peptides

At present,the excessive use of antibiotics is increasing the number of multi-resistant bacterial strains,which poses a great threat to human health and public health.Therefore,the discovery and production of new antibacterial agents have more and more important significance.Antimicrobial peptides(AMPs),as natural immune active substances in organisms,can effectively avoid the generation of multiple drug resistance of bacteria.Due to the difficulty in isolation and extraction of natural AMPs,current strategies to produce AMPs using recombinant DNA techniques usually involve affinity chromatography and proteases,which are cumbersome and expensive.To solve these problems,the ELP-intein self-cleavage system without chromatography and protease was used to prepare AMPs in this study.The fusion protein was purified by the reversible phase transformation behavior of ELP(elastin like polypeptide)with temperature,and the target AMPs were obtained by the C-cleavage of the p H-sensitive Mtu Rec A ?I-CM intein.Four AMPs were selected through literature review: arasin-like Sp,Brevinin-2,HD-5 and LCNKL2.As an aquatic AMP,arasin-like Sp had good antibacterial activity against aquatic pathogens such as Vibrio harveyi(V.harveyi).While Brevinin-2,HD-5 and LCNKL2 could effectively inhibit Staphylococcus aureus(S.aureus)and Escherichia coli(E.coli).After synthesis,the genes were cloned into expression vector containing ELP and Mtu Rec A ?I-CM intein,respectively.The recombinant expression plasmids p ET-ELP-I-arasin-like Sp,p ET-ELP-I-Brevinin-2,p ET-ELP-I-HD-5 and p ET-ELP-I-LCNKL2 were constructed.In E.coli as host cells,fusion proteins were expressed in soluble form.In the end,arasin-like Sp,Brevinin-2 and HD-5 were successfully obtained by ELP purification and C-cleavage of the intein,with yields of ? 4 mg/L,? 5mg/L and ? 4.6 mg/L,respectively.The antibacterial experiments showed that purified arasin-like Sp,Brevinin-2 and HD-5 had antibacterial activities.FE-SEM and PI staining analysis showed that they could change the morphology and membrane permeability of bacteria.Hemolysis test results showed that arasin-like Sp,Brevinin-2 and HD-5 all showed low hemolysis in mouse erythrocytes,indicating that AMPs produced using this system have the potential to become an effective substitute for antibiotics in the future.In this method,the target AMPs could be obtained only through simple ammonium sulfate precipitation and p H change,and the purified AMPs retained their original activity,which was efficient and cost saving,and laid a foundation for the large-scale production of AMPs.At the same time,because AMPs have good antibacterial activity and anti-biofilm effect,it indicates potentials for the treatment of microbial infection caused by postoperative or other wounds.To expand the application of AMPs in this field,the fusion of AMPs and spider silk protein was developed as a new drug free material containing AMPs to prevent or reduce the risk of infection.Based on the study of spider silk fiber in our laboratory,the above-mentioned HD-5 was cloned into the downstream of Flag-Ac Sp1(FAc),a chimeric spider silk protein with good mechanical properties,and separated by the thrombin site.After being expressed in E.coli,the recombinant protein was located in the supernatant.The recombinant protein was purified by Ni-NTA agrose under native condition,and the mechanical properties of the recombinant protein silk fiber were measured after being prepared by manual drawing.The results showed that,although the recombinant protein 6×His-SUMO-FAc-HD-5 fiber did not possess the excellent stress and Young's modulus of FAc fiber,it kept the good toughness of FAc fiber and showed excellent strain capacity.HD-5 was obtained after thrombin digestion and Ni-NTA agrose purification again.The antibacterial activities of recombinant proteins 6×His-SUMO-FAc-HD-5 and HD-5 were determined,and the results showed that they formed antibacterial circles against S.aureus and E.coli,which to some extent indicated that the AMPs purified by Ni-NTA agrose had antibacterial activities.In conclusion,we successfully obtained AMPs from E.coli using the ELP-intein self-cleavage system,providing an economical and effective way for large-scale production of AMPs in the future.In addition,chimeric spidroin-HD-5 recombinant protein was successfully expressed in E.coli,which laid foundation for the further study of AMPs as antibacterial coatings and wound dressings.