Expression And Purification Of The Targeting Fusion Proteins Of Several Antitumor Antimicrobial Peptides

Pc-pis is a piscidin-like antimicrobial peptide(AMP)derived from big yellow croaker.In previous studies,we found that Pc-pis can rapidly kill tumor cells.In order to develop its application in antitumor,we conducted preliminary research in the following aspects.HeLa cells were treated with AMP Pc-pis,and the morphological characteristics of cell death were observed by SEM.There are a lot of curved villi on the surface of normal cells membrane which is smooth and complete.The treatment of Pc-pis made the cell shape round,The number of the villi decreased,and the holes appeared on the membrane.It shows that Pc-pis causes cell death by changing the structure of cell membrane.AMP could be applied to molecular targeted therapy by the use of cell-type specific binding due to ligand/receptor interaction.Mesothelin/MUC 16,urokinase/receptor and CD 47/SIRPa were selected as targeted delivery system.It was proved that these three pairs of molecules could interact with Co-IP,respectively.In previous work,we found that the Piscidin 1,Piscidin 3,and Hb 26(pleurocidin family)have a similar killing effect on cancer cell as Pc-pis.Because AMP are toxic,it is difficult to be expressed by prokaryotes,so the expression and purification of fusion proteins of antimicrobial peptides have been the focus of research in this field.We designed a fusion protein backbone and linked AMP to an expression system consisting of GST purification tags,antibody hinge regions,Thrombin,His tag,and targeting ligand to target AMP to the molecules on the surface of tumor cells.We explored the expression conditions successfully.The fusion protein containing the corresponding ligand/receptor and AMP was obtained by two-step purification,which provides a certain basis for the development of clinical application of AMP.