Construction Of Prokaryotic And Eukaryotic Secretory Expression System For Cationic Antimicrobial Peptides

Cationic antimicrobial peptides are small molecule polypeptides with widely antimicrobial activity. Cationic antimicrobial peptides are evolutionarily conserved and important innate immune molecules.Because antimicrobial peptides are limited from natural sources, chemical synthesis is expensive; however antimicrobial peptides producing by genetic engineering technology have a high yield, strong activity and donnot waste much time; this method has become the ideal way to produce antimicrobial peptides currently.Cationic antimicrobial peptides are toxic to E. coli and susceptible to degraded by protease, therefore we add a fusion partner to N-terminus of the antimicrobial peptide which containing negative charges to neutralize positive charge of the antimicrobial peptide. The cleavage of fusion partner is an important step after the fusion expression of antimicrobial peptides. It is high costly using enzyme to cleave it. In chemical cleavage method the CNBr is toxic to human and environment,so this method is difficult to use widely. In this experiment we introduce Asp-Pro acid-sensitive sites between the fusion partner and antimicrobial peptide, using acid to cleave fusion peptide.This method is easy to operate and cheap. The fusion partner used in the experiment is Cherry which may promote the fusion protein secreted into the culture supernatant however it cannot guarantee completely secreted. We add glycine during the induction in this study, which can interfere with the synthesis of peptidoglycan layer of cell wall and destroy the integrity of the cell membrane, resulting the release of protein in the periplasmic space including the fusion protein and increasing the secretion efficiency finally.Pleurocidin is isolated from the skin mucus of winter flounder (Pseudopleuronectes americanus) which is a linear polypeptide containing 25 amino acid residues and has a broad-spectrum antimicrobial activity and can inhibit a variety of Gram-positive and Gram-negative bacteria and fungi. We ligate the gene expresses Pleurocidin and Cherry DNA sequence via blunt tail.Then we clone this fusion gene to pET22b (+) vector and transforme recombinant plasmid into E. coli BL21 (DE3). We use lactose(final concentration is 4 g/L) to induce the expression fusion protein and the induction time is 20 h.The fusion protein express successfully.We add glycine (final concentration is 4 g/L) after 16 h of induction, almost all fusion protein secreted into the culture supernatant.The production of fusion protein reached 516 μg/mL. The Asp-Pro acid sensitive site locate between Cherry tail and Pleurocidin gene, add dilute hydrochloric acid to the culture supernatant to the final concentration of hydrochloric acid is 100mM, the hydrolysis tempreture is 65 ℃ and the hydrolysis time is 72 h.We can obtain the active Pleurocidin using this hydrolysis method. Pleurocidin has significant antibacterial activity to E. coli DH5a and Staphylococcus aureus.Bovine lactoferrin peptide (LfcinB)is a domain released from the N-terminal of bovine lactoferrin which can be hydrolyzed by stomach in the acidic environment. LfcinB contains 25 amino acid residues and has a wide and efficient antibacterial activity.In this experiment, we also study the secretion expression of antimicrobial peptide in Saccharomyces cerevisiae. We substitute the GAL1 promoter of pYES2 plasmid using a constitutive promoter TPI. We construct pYES2-TPI-LfcinB recombinant plasmid successfully and electroplate Saccharomyces cerevisiae INVScl. We screen positive clones by medium for yeast without uracil(SC-U medium) and obtaine Saccharomyces cerevisiae pYES2-TPI-LfcinB strains. This strain does not need induce in the training process and LfcinB can secretory express directly. This strain does not exis the problemt of glucose inhibition the expression of LfcinB. make The antibacterial experiments of culture supernatant show:LfcinB has significant antibacterial activity to E.coli and Staphylococcus aureus. This experiment provide technical support to antimicrobial peptides constitutively secretory expression in Saccharomyces cerevisiae and has a certain guiding significance.