Fusion Expression In E.coli, Purification And Activity Research Of A New Antimicrobial Peptide 'CN20' In Breast Milk

With the study of peptide group is becoming a hot spot of recent research and the breast milk in infant healthy growth and the huge function of disease prevention, the study of antibacterial peptides in breast milk is significant. However, it's difficult to estimate the cost to isolate antimicrobial peptides from the body or in breast milk directly. In this study,we isolateted endogenous peptides from colostrum of premature mothers and the antimicrobial activity was detected. Then we predicted an antimicrobial peptide from the results of mass spectrum. We named it 'CN20' because it comes from ?-casein. Antimicrobial experiments improved the antimicrobial activity of CN20 which comes from chemical synthesis.In order to obtain a large number of CN20 proteins while reducing costs,we cloned the gene of CN20 into pET32a(+) vector. Then the plasmid was transformed into E.coli BL21(DE3) to express CN20 proteins. After optimization of induction conditions such as inducer concentrations, induction tempreture, time and speed. We got an optimum contion for expression. We purified CN20 by nickel colomn affinity chromatography because there is a 'His-tag' at the C end. Western blot can detect the fusion protein. We added a TEV enzyme cutting site between trxA tag and CN20 so that we can get CN20 protein without trxA tag by TEV enzyme. Finally, a series of antimicrobial experiments showed activity of CN20 and we established its antimicrobial spectrum. We also studied about relationship between antimicrobial activity and reaction time.To sum up,a new antimicrobial peptide was isolated from the breast milk and successfully expressed in E.coli which detects the corresponding antimicrobial activity. This study may provide the theoretical and experimental basis for further researches of recombinant expression of antimicrobial peptides and the development of new drugs.