In order to have more efficient to get our AMPs, we treated signal peptide upstream of the N-terminal of antimicrobial peptides to facilitate the peptide production. This signal peptide is comes from chitinase C of S.lividans (MGFRHKAAALAATLALPLAGLVGLASPAQA).[1] After translation process signal peptide will lead AMPs to the secretion system of E.coli. [2] When the pre-mature peptides go through the periplasmic space, peptidase will identified the cleavage site Ala-Gln-Ala and cut at the double Ala at the signal and mature peptide linkage site. Then separate signal peptide from AMPs. Finally, secreting AMPs to the LB culture medium.

To make sure the secretion system is work we attached an Ala at the N-terminal of AMPs. So we had modified the amino acid sequence of Signiferin and Epinecidin-1 to facilitate the secretion process. And we had used structure prediction software to analysis the attached Ala affect the peptide folding process or not in our modeling page.