Design And Expression Of The Hybrid Antimicrobial Peptides CL23 And LH28

The emergence of antibiotic resistance issues, which pose a great threat to the human health, demand that new antimicrobial preparations should be explored for treating these infections. Antimicrobial peptides not only have direct antimicrobial activity but also are innate immune regulators, which possess of the potential as beneficial therapeutics in overcoming infectious diseases.The method that aims to optimize the design of hybrid peptides and screen the most potential ones based on the structure and activity relationship of antimicrobial peptides by bioinformatics tools was tested. Bovine lactoferricin analogue LFB15-W4,15, Cecropin A and HP(2-20) were picked out as the parent peptides, and intercept different fragments of parent peptides to design hybrid peptides, in order to obtain ones with higher antimicrobial activity and lower hemolysis. With bioinformatics tools, physical-chemical parameters of hybrid peptides were calculated, the secondary structure were predicted and the parameters were optimized. Two potential hybrid peptides: CA(1-8)-LFB15W(4,10),abbreviated as CL23 and LFB15(W4,10)-HP(4-16) , abbreviated as LH28, were chosen for synthesis. The antimicrobial and hemolytic assays were carried out to test the rationality of the design. Hybrid peptides had boarder antimicrobial spectrum and stronger antimicrobial activity than their parent peptides. The concentration produced low hemolysis was higher than MIC; the antimicrobial activity against E.coli is four times higher than parent peptide LFB15-W4,10 ;The results demonstrated that optimization of the parameters based on structure-activity relationship of antimicrobial peptides are powerful methods for molecular design of antimicrobial peptides. Results demonstrated that amphipathicity is the key structural feature that influences the antimicrobial activity; high hydrophobicity enhances the hemolytic activity of the antimicrobial peptides.The method for setting up high efficient E.coli recombinant expression system was tested. Expression as Monomer and polymer, the toxicity tolerant and expression enhancing effect of different fusion protein tag; cleavage of fusion protein using enzymatic and chemical methods, the toxicity tolerance of different host strains, were compared and researched, which influence the high efficient expression and following purification. First, expression of two hybrid peptides as fusion protein in E.coli BL21(DE3). According to the E.coli codon bias, their genes were synthesized and the cleavage sites of EcoR I, Not I and factor Xa cleavage sites coding sequence were added to the terminals of the genes, the genes were inserted into the vectors: pET28a(+), pET32a(+) and pET41a(+). Six recombinant plasmids were successfully constructed. After transformed and induced by IPTG, fusion protein of recombinant pET28a expression vector was not detected by SDS-PAGE. Fusion protein expressed as inclusion body from recombinant pET41a vector. Fusion protein expressed in solvent form from recombinant pET32a vector, which were purified by Ni chromatography. The purity of fusion protein was about 90%, but factor Xa cleavage specification was not effective enough and it caused great difficulties for following purification. Amplifying the culture volume will increase the usage of the enzyme, which increases the cost of production.Base on analysis of the moner expression experiment, LH28 was chosen for the polymer expression research which aims to enhance the proportion of the target peptides in fusion protein and the ratio of obtaining the peptides. Formic acid and hydroxylamine cleavage sites were inserted between multimer and vector protein and among peptide monomers. According to the codon bias of E.coli, monomer genes were synthesized and inserted into plasmid pUC19 using KpnI and HindIII. The end of the gene had BbsI cleavage sites for constructing the recombinant pUC19-(LH28)n , and pUC19- (LH28)2-6,8 were correctly constructed. Tandem genes were inserted into pET32a using KpnI and HindⅢand the recombinant vectors pET32a-(LH28)2-6,8 were correctly constructed. IPTG induction caused OD600nm of BL21(DE3) culture decreased obviously while C41(DE3) and C43(DE3) had no OD600nm decreasing phenomenon, which had higher toxicity tolerance than BL21(DE3). Except dimmer fusion protein, Trimer to hexamer and octamer fusion protein could not be detected on the SDS-PAGE, Under the same induction conditions, the biomass (OD600nm)of C43(DE3) was the highest and SDS-PAGE results demonstrated that dimer fusion protein showed the highest expression level in C43(DE3), about 35% of total cell proteins. After Ni chromatography purification, 63 mg fusion protein Trx-(LH28)2 with purity above 95 % was obtained from 1 litre of E. coli culture. The LH28 dimer fusion protein was effectively cleaved by 50 % formic acid at 50℃after 36 h. After Ni chromatography purification, the purity of recombinant LH28 (P-LH28-D) is aboat 85 % and P-LH28-D displayed almost the same antimicrobial activity against B.subtilis and S.aureous with the synthesized peptide LH28.The results demonstrated that the method that using bioinformatics tools to optimize the design of hybrid peptides and screen the most potential hybrid peptides has instructive effect on the design of the hybrid peptide and save the cost and time, hybrid peptides CL23 andLH28 can be used as the models for design new antimicrobial peptides.In this study, fusion protein of LH28 dimer were expressed in the host C43(DE3), most Trx-(LH28)2 was expressed as inclusion body, which facilitate the following purification; Formic acid can solve the water-insolvent protein, which avoid refolding protein and wiping off the enzyme, as well as lower the cost. This system lays a foundation for the expression and purification of the other hybrid peptides.