Prokaryotic Expression And Functional Verification Of Antimicrobial Peptide LR_GG

Antibacterial peptide is a kind of small molecule polypeptide with antibacterial activity.Because of its strong antibacterial activity and low toxicity,it has a wide application prospect in the clinical market.However,due to the low content of antimicrobial peptides in organisms and the complex purification process,the yield of biological extraction cannot meet the clinical requirements.However,although chemical synthesis can quickly obtain antimicrobial peptides,it is difficult to realize industrialization because of the high cost,resulting in a small number of clinical antimicrobial peptides that have been marketed.E.coli expression system is the most widely used expression system in the field of genetic engineering.It has the advantages of clear genetic map,simple operation,short expression cycle and high yield.Given the excellent properties of the E.coli expression system,In this study,we used the gene engineering method to express the antimicrobial peptide LR_GG antagonizing Gram-negative bacteria.In order to achieve the purpose of quantitative production of antimicrobial peptides,to provide theoretical reference for subsequent clinical application.The results are as follows:In this study,fusion protein expression vector p QE-GFP-LR_GG was successfully constructed,fusion protein GFP-LR_GG with molecular weight of about 31 k Da was obtained by IPTG induced expression and nickel column purification.The molecular weight of LR_GG was about 1.9 k Da,and its size was consistent with the theoretical value.（1）The optimal expression conditions of fusion protein GFP-LR_GG were:E.coli was cultured in 2×YT medium to logarithmic growth stage.add 1.2m M IPTG,It was induced at 37.27℃for 4.18 h.Under these conditions,the yield of fusion protein GFP-LR_GG was increased by35.6%.（2）The prokaryotic peptide LR_GG showed a broad spectrum of antibacterial activity against Gram-negative bacteria,killing more than 99.99 percent of E.coli ATCC25922 within 100min.At the same time,the prokaryotic expression of LR_GG has good temperature,p H,salt ion and serum stability,which is basically the same as the chemical synthesis of LR_GG.（3）Both prokaryotic expression and chemical synthesis of LR_GG were cell selective and had no embryotoxicity,The prokaryotic peptide LR_GG showed good therapeutic effect on the infection model of C.mellonalis.（4）The prokaryotic peptide LR_GG can cause the death of Gram-negative bacteria by destroying their cell membranes.In summary,this study successfully expressed antimicrobial peptide LR_GG using prokaryotic expression system,The production of LR_GG was increased,and the expression of LR_GG still had strong antibacterial activity and maintained the original physiological and biochemical properties.The research will lay a foundation for the wide application of antimicrobial peptides.