Recombinant Expression of a Plant-Derived Dimeric Antifungal Peptide (DiSkh-AMP۱) Joined by a Flexible Linker in Escherichia coli and Evaluation of Its Biological Activity in-Vitro

BACKGROUND AND OBJECTIVESDiSkh-AMP۱, a novel dimeric antifungal peptide contained ۶۵ amino acid residues was recombinant produced by a flexible linker to improve the antifungal activity of native Skh-AMP۱ isolated from Satureja khuzistanica leaves.MATERIALS AND METHODSTo discover a new system for expressing DiSkh-AMP۱ in Escherichia coli BL۲۱, the DiSkh-AMP۱ gene was synthesized and inserted into pET۲۸a vector in which fusion peptide (۳.۵ kDa) was used as a fusion partner and an N-terminal ۶-His as an affinity tag. The fusion/DiSkh-AMP۱ peptide expression was induced with ۱ mM isopropyl-thio-galactoside (IPTG) for ۴ h at ۳۷ ºC. The recombinant fusion/DiSkh-AMP۱ was then purified by affinity chromatography, digested with cyanogen bromide and isolated from the fusion peptide by reverse-phase high-performance liquid chromatography.RESULTS AND DISCUSSIONThe final yield of DiSkh-AMP۱ was ۰.۹۵ mg/L after cleavage with cyanogen bromide. DiSkh-AMP۱ strongly inhibited the growth of Candida albicans (ATCC ۱۰۲۳۱) and Aspergillus fumigatus (Af ۲۹) by MIC values ۶.۵ and ۶.۸۶ μM respectively. It showed fungicidal activity against Candida albicans (ATCC ۱۰۲۳۱) and Aspergillus fumigatus (Af ۲۹) with MFC values ۱۳.۰۰ and ۱۳.۷۲ μM respectively. DiSkh-AMP۱ had a negligible hemolytic activity of ۴.۱% for human red blood cells.CONCLUSIONTaken together, our results demonstrated that DiSkh-AMP۱ with improved antifungal activity and suitable yield in expression system could be considered as a potential candidate to be a novel antifungal drug against life-threatening fungal infections.

کلیدواژه ها:

Skh-AMP۱ · Dimeric antifungal peptide ، Candida albicans ، Aspergillus species ، Flexible linker ، Recombinant expression ، Escherichia coli.