Construction And Application Of A Carrier Protein-mediated PR-39 Antimicrobial Peptide Expression System

Objective:Proline-rich antimicrobial peptides(Pr AMPs)kill bacteria by a non-cleavage mechanism,which have a low propensity for microbial resistance,and may be new candidates for peptide antibiotics.Porcine PR-39 antimicrobial peptide is derived from porcine neutrophils or other immune cells,and is an antimicrobial peptide of the cathelicidin family containing 39 amino acid residues.It has been found that PR-39 can be isolated and purified from porcine small intestine tissue as well as peripheral blood neutrophils,and has broad-spectrum antibacterial activity against common pathogenic microorganisms,as well as immune functions such as chemotaxis,inhibition of inflammation,and promotion of damage repair.The present study was conducted to construct a suitable molecular expression system for PR-39,which is of great importance to solve the problem of low expression and poor stability of Pr AMPs.Methods:In this study,we searched the literature for carrier proteins that might carry PR-39 expression,at the mean time,we determined that Saccharomyces cerevisiae β-(1,3)-glucanase Exg1 p and chicken-derived Ca Modulin(Ca M)could be secreted and expressed in pichia pastoris by combining protein data and expression analysis.The proline-rich antimicrobial peptide PR-39 was designed to be replaced in the flexible loop region at the N-terminal end of Exg1 p and attached to the C-terminal end of Ca M,respectively,to mediate the expression and purification of PR-39 in P.pastoria strains GS115 by Exg1 p and Ca M,and to examine the relevant antimicrobial activity of PR-39.Results:The results showed that Exg1 p and Ca M could mediate the expression of PR-39 in P.aeruginosa GS115,and the yield of Ca M-PR-39 in shake flask culture could reach about 1.0-1.2 mg/L.In addition,Ca M-PR-39 could be obtained as a single pure product after enzymatic digestion on a nickel ion affinity chromatography column,which is a simple and time-consuming purification method.This method is simple and timeconsuming,and the design provides a reference for the fusion expression and purification of other proteins of the same type.In addition,we examined the antibacterial activity of PR-39 by tenfold dilution counting method and also measured the growth curves of different strains of bacteria,and found that the Ca M-mediated expression of PR-39 had good antibacterial and bactericidal activities against E.coli.Conculsion:This study identified Exg1 p and Ca M as heterologous carrier proteins for expressing antimicrobial peptides,and developed a Ca M-based fusion protein bioexpression platform to provide a new technical strategy for the preparation of PR-39 and other antimicrobial peptides.