Molecular Desigh Of The Hybrid Antimicrobial Peptide Of LFM-3and Its Expression In Pichia Pastoris

With the overuse of antibiotics, it leads to enormous selection pressure for the pathogens, which causes the emergence of many antibiotic resistant strains and results in drug residues in food which endangers people’s health directly and indirectly. Antimicrobial peptides are a class of the earliest produced immune active molecules by organisms in evolution process, the rather ancient composition in animal innate immune response process, and very important in animal innate defensive system. The antimicrobial peptides, consisting of20-100amino acid residues, widely exist in plants and animals and microorganisms, and have advantages of broad-spectrum antimicrobial activity and not easily form resistance force. Antimicrobial peptides not only have anti-bacterial, anti-virus, anti-tumor, and anti-parasitic function, but also role in immune regulation, cell signaling, cell proliferation regulating and other aspects. Therefore, antimicrobial peptides have broad application prospects in clinical medicine and animal health. However, compared with antibiotics, natural antimicrobial peptides have relatively narrow antibacterial spectrum, high cost of synthesis, and some of them have a hemolysis effect on eukaryotic cells. Thus, on the method of molecular design and modification, it is very meanful to develop a kind of antimicrobial peptides that have higher antimicrobial activity and lower hemolysis effect.Though there is still a lot of difficult in molecular design of antimicrobial peptides, it is yet possible to filter out a kind of much better antimicrobial peptides by using modern bioinformatics methods which include calculating the structural parameters and analyzing the impact of these structural parameters on the activity of the designed antimicrobial peptides. It is one of the most effective methods of acquiring a sort of much better antimicrobial peptides that the core sequences of different antimicrobial peptides were chosen to combine new peptides with the modern bioinformatics tools. On this study, the two sequences of bovine lactoferricin and Magainin-2were picked out as the parent peptides, and intercept different fragments of parent peptides to have designed six a-helix hybrid peptides. With bioinformatics tools, the physical-chemical parameters of them were calculated. Their net positive charge, hydrophobic force, amphiphilic strength, and helicity,were compared. Then, the helical wheel maps were drawn to analyze the features of the distribution of the amino acid and charges for the six hybrid peptides. After that, we sent the sequences of the six peptides to company to be synthesized, and the MIC and hemolysis effect of them were detected. The results showed that the antimicrobial activity of the synthetic hybrid peptide LFM-3had the strongest antimicrobial activity of the six peptides. The MIC of the LFM-3was16μg/mL against gram-positive Staphylococcus aureus and32μg/mL against gram-negative E. coli. In additon, high concentrations of LFM-3had slight hemolytic effect on chicken’s red blood cells.According to the amino acid sequence of the LFM-3and the Pichia pastoris preferred codons, the base sequence of the target gene was determined. The synthesized gene was subcloned into the shuttle vector pPICZaA after being digested with the two restriction endonuclease Xba I and Xho. The constructed vector LFM-3-pPICZαA was transformed into E. coli DH5a. A positive colony was picked to zoom in medium, and then a large number of recombinant plasmids was extracted and purified by large-scale alkaline lysis method. The recombinant plasmids were linearized with single restriction enzyme Sac I, then was transformed into host Pichia pastoris (SMD1168) by electroporation method. When the recombinant colonies grew at the diameter of2millimeter, they were transferred to selected YPDS plates which contained200μg/mL,500μg/mL, and1000μg/mL Zeocin, respectively. The recombinants that could normally grow at the highest concentration of Zeocin were considered to be high copies transformed strain. After being induced expression with methanol in flasks, the antimicrobial activity of its supernatant was initially detected. It showed that the diameter of the inhibition zone against Staphylococcus aureus Cowan I and E. coli K88reached to20mm and17mm, respectively, whereas the supernatant of the negative control of the empty vector expression showed no inhibited activity. In search of the inducing conditions in flasks, we found that, when the recombiant yeast was induced for72hours, with started OD600between2～4, temperature at28℃, inducing methol concentration of0.5%and starting pH at6.0, the expression level reached to the highest point. We have maken many efforts on the purification process of the expressed antimicrobial peptide, including dialysis and concentrating to the expresse supernatant, trying defferent binding buffer and elusive buffer, using all kinds of chomatography media, and so on, but the detection results always showed that the expressed antimicrobial peptide was not able to combine with all the media. We speculated that the expressed antimicrobial peptide might have a high unit activity but was at a low expressing level somehow.