Recombinant Expression of a Plant-Derived Dimeric Antifungal Peptide (DiSkh-AMP1) Joined by a Flexible Linker in Escherichia coli and Evaluation of Its Biological Activity In Vitro

DiSkh-AMP1, a novel dimeric antifungal peptide contained 65 amino acid residues was recombinant produced by a flexible linker to improve the antifungal activity of native Skh-AMP1 isolated from Satureja khuzistanica leaves. To discover a new system for expressing DiSkh-AMP1 in Escherichia coli BL21, the DiSkh-AMP1 gene was synthesized and inserted into pET28a vector in which fusion peptide (3.5 kDa) was used as a fusion partner and an N-terminal 6-His as an affinity tag. The fusion/DiSkh-AMP1 peptide expression was induced with 1 mM isopropyl-thio-galactoside (IPTG) for 4 h at 37 ºC. The recombinant fusion/DiSkh-AMP1 was then purified by affinity chromatography, digested with cyanogen bromide and isolated from the fusion peptide by reverse-phase high-performance liquid chromatography. The final yield of DiSkh-AMP1 was 0.95 mg/L after cleavage with cyanogen bromide. DiSkh-AMP1 strongly inhibited the growth of Candida albicans and Aspergillus species by MIC values from 6.5 to 7.22 µM. It showed fungicidal activity against tested fungi with MFC values from 13.00 to 14.45 µM. DiSkh-AMP1 had a negligible hemolytic activity of 4.1% for human red blood cells. Taken together, our results demonstrated that DiSkh-AMP1 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.