Bioproduction and Characterization of Epinecidin-1 and Its Variants Against Multi Drug Resistant Bacteria Through In Silico and In Vitro Studies

The antimicrobial peptide Epinecidin-1 (Epi-1) obtained from the marine grouper fish ( Epinephelus coioides ) possessing a variety of antibacterial, anticancer, and anti-inflammatory effects, led us to choose this peptide an ideal candidate for enhancing its therapeutical potential and biosynthesis by recombinant DNA technology. In the present study, we improved the therapeutic value of Epi-1 by replacing with lysine amino acid at certain positions based on proven bio-informatics dogma, named the improved peptides as Variant-1 and Variant-2 and produced by genetic engineering method. After designing the peptides for the desired effect, we proceeded for recombinant production by cloning their individual genes in-frame with a thioredoxin fusion partner in the pET32a vector background. The genes for Epi-1 and its variants were created by overlap extension PCR. The fusion antimicrobial proteins were evaluated for optimized over-expression, purification and digested with enterokinase to release active peptide fragment. Antibacterial activity of the fusion protein and enterokinase digested fusion antimicrobial peptide fragments were evaluated, which resulted in enhanced antibacterial activity against tested multi drug resistance strains of Gram- negative Escherichia coli (MTCC 2939), Pseudomonas aeruginosa (MTCC 741), Klebsiella pneumoniae (MTCC 432) and Gram-positive Staphylococcus aureus (ATCC 25923).