Year: 2026
Type: preprint
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.
Source: Research Square
Institution: Kashi University
Language: English
Subfield: Microbiology
Field: Immunology and Microbiology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W7225206282