Design, Dimerization, and Recombinant Expression of a Plant‑Derived Dimeric Antifungal Peptide (DiMCh-AMP۱) in Escherichia coli and Evaluation of Its Biological Activity in Vitro

BACKGROUND AND OBJECTIVESFungal species resistant to current antifungal agents are considered as a serious threat to humanhealth, the dilemma that has dragged attentions toward other sources of antifungals such asantimicrobial peptides (AMPs).MATERIALS AND METHODSIn order to improve biological activity of a recently described antifungal peptide MCh-AMP۱from Matricaria chamomilla flowers, MCh-AMP۱dimer (DiMCh-AMP۱), containing ۶۱amino acid residues connected by flexible linker (GPDGSGPDESGPDES), was designed andexpressed in Escherichia coli, and its structure was analyzed using bioinformatics tools.DiMCh-AMP۱ synthetic gene was cloned into pET-۲۸a expression vector, which was then usedto transform E. coli BL۲۱ (DE۳) strain. His-tag purification was achieved using metal-chelateaffinity chromatography. Because there is no methionine residue in the DiMCh-AMP۱sequence, cyanogen bromide was successfully used to separate the target product from the tag.Reverse-phase high-performance liquid chromatography was used as the final step ofpurificationRESULTS AND DISCUSSIONResults showed that recombinant peptide was produced in considerable amounts (۰.۹ mg/L)with improved antifungal activity toward both yeasts and molds compared to its monomericcounterpart. The minimum inhibition concentration and minimum fungicidal concentrationvalues of DiMCh-AMP۱ against Candida and Aspergillus species were reported in the rangeof ۱.۶۷–۶.۶۶μM and ۳.۳۳–۲۶.۶۴μM, respectively.CONCLUSIONOur results showed that while antifungal activity of dimerized peptide was improvedconsiderably, its cytotoxicity was decreased, implying that DiMCh-AMP۱ could be a potentialcandidate to design an effective antifungal agent against pathogenic yeasts and molds.

Keywords:

Antifungal Activity , Cytotoxicity , Antimicrobial Peptide , Aspergillus , Candida , Bacterial Expression System