Figure 2. The vector map of the r5M-172-AMP-173 expression construct and the production strategy of antimicrobial peptides using r5M-GFP system in E. coli.

(a) AMPs were inserted between 172 and 173 amino acids positions of r5M-GFP. Loop structure forming linker sequences, GGSGT and GSGG, are used for cloning and indicated in amino acids. The letter “M” flanking both 5′ and 3′ side of AMP indicates methionine. The restriction sites and 6x His-tag at both N- and C-terminals are indicated. (b) AMPs are cloned into a loop region of methionine-free GFP (r5M-172-AMP-173) and expressed as insoluble proteins in E. coli. The insoluble proteins are extracted and purified by Ni-NTA and cleaved at methionine sites using cyanogen bromide. Finally, AMPs are purified by high performance liquid chromatography.