SNAC-Mediated Antimicrobial Peptide Production: Comparison with Enterokinase Digestion

Abstract Antimicrobial peptides (AMPs) are promising alternatives to conventional antibiotics, but their small size and host cytotoxicity hinder direct recombinant production. In this study, six AMP genes (LK-24, GSHHW-LK-24, ZY4, GSHHW-ZY4, Mast-MO and GSHHW-Mast-MO) were synthesized by primer-complementary PCR and cloned into pET32a, generating two series of vectors that carry either the enterokinase recognition site DDDDK or the sequence-specific nickel-assisted cleavage (SNAC) tag GSHHW. The fusion proteins were expressed in Escherichia coli BL21(DE3) by IPTG induction and auto-induction, purified from the soluble fraction by Ni-NTA affinity chromatography, and cleaved by enterokinase digestion or SNAC-mediated chemical cleavage. Antibacterial activity was evaluated by zone-of-inhibition and minimum inhibitory concentration (MIC) assays against E. coli ATCC 8739 and Staphylococcus aureus ATCC 25923. All six AMPs were active, with inhibition zone diameters of 6.0-9.5 mm and MIC values of 4–32 µg/mL. The peptides released by SNAC cleavage gave slightly larger inhibition zones (7.0-9.5 mm) and two-fold lower MIC values (4–16 µg/mL) than those released by enterokinase digestion (6.0-8.5 mm; 8–32 µg/mL), although the differences were not statistically significant. SNAC-tag chemical cleavage therefore provides a cost-effective, protease-free strategy for the efficient preparation of recombinant AMPs.