Exploring The Expression Of Antibacterial Peptide Targeting Fusion Proteins

Piscidin series and its similar antibacterial peptides are conservative linear short peptides of amphiphilic,positively charged marine fish.The five AMPs we selected,pc-pis,Piscidin,Piscidinl,Piscidin3 and Hb26,all had anti-tumor activity except Piscidin,and were good anti-tumor antimicrobial peptides.In order to study its anti-tumor mechanism and enhance targeting,we use ligand/receptor or single-chain antibody/antigen interactions to construct a specific fusion protein that targets cancer cells and explore its prokaryotic expression.In order to study its antitumor mechanism,we constructed AMP series of prokaryotic expression vectors,all of which can be soluble expressed in E.coli.The labeled AMP series proteins were incubated with the general lipid strip,respectively.Western blot analysis showed that GST-AMP series,which has antitumor activity,can bind to most negatively charged membrane phospholipids.Due to GST-ligand/single chain antibody-Pc-pis could not solublely express in E.coli,we modified Pc-pis C terminal with calmodulin(Calm2),the results showed that GST-ligand/single antibody-Pc-pis-Calm2 could solublely express,Pc-pis the N terminal moedfication with Calm2 were not soluble expression in E.coli,but to add a proper length of hingeafter Calm2 or GST,we found thatit can promote the solubleexpression.The c-terminal is the AMP active terminal,and the residual amino acids after Calm2resection may affect its function.In order to facilitate purification and minimize unnecessary modification,a two-step purification recombinant plasmid for prokaryotic expression was constructed.Single-chain antibody proteins are mainly her2-targeting 4D5scFv,EGFR-targetingy F4 and EpCAM C215.Some recombinant plasmids were found to be prokaryotic soluble expressions such as 4D5scFv?AMP and C215?AMP.The protein bands can also be seen after enzyme digestion.However,F4-AMPseemed not to be soluble expression.In a word,we expressed and purified several targeted fusion proteins of AMPs andwish that they could be delivered to specific cancer cell surfaces to kill the cells,which will enhance specificity,reduce potential side effects.Our results provided theoretical basis for applications of antitumor AMPs.