Abstract:[Objective] The antimicrobial peptide, YFGAP from yellowfin tuna (Thunnus albacores), consisted of 32 amino acid residues, shows broad-spectrum antimicrobial activity against Gram-positive and Gram-negative bacteria without hemolytic activity. Clone and express the antimicrobial peptides, YFGAP in Escherichia coli BL21(DE3) and characterize its bioactivity. [Methods] Based on the amino acids of EK-YFGAP and L-EK-YFGAP, the genes were synthesized and ligated into pET-22b to construct these expression vectors, pET22b-ELP20-EK-YFGAP, pET22b-ELP40-EK-YFGAP and pET22b-ELP40-L-EK-YFGAP. The vectors were transformed into E. coli BL21(DE3) to express the fusion proteins. The fusion proteins were purified using the Elastin-like polypeptides (ELPs) as a purification tag by the method called inverse transition cycling (ITC) and digested by enterokinase. Then the recombinant YFGAP was purified using Vivaspin Turbo. Bioactivity of the peptide was tested. [Results] The fusion proteins, ELP40-EK-YFGAP and ELP40-L-EK-YFGAP were expressed and purified by two rounds of ITC. After digested by enterokinase, the recombinant YFGAP was purified using Vivaspin Turbo. Antimicrobial activity assay demonstrated the recombinant YFGAP exhibited high antibacterial activity against four fish pathogens without significant hemolytic activity. [Conclusion] The recombinant YFGAP was successfully expressed in E. coli using ELPs as a purification tag. This strategy does not require the use of chromatography, so that it is cost effective, easy to scale up and to multiplex. This study provides theoretical foundation and technical means for scale-up preparation of antimicrobial peptides by engineering method.
