Comparative Analysis of Three Preparation Strategies for AntiMicrobial Peptides and Their Antibacterial Efficacy

Antimicrobial peptides (AMPs) are regarded as promising candidates for the post-antibiotic era because of their broad-spectrum bactericidal activity and low propensity to induce bacterial resistance. However, their small molecular weight and intrinsic cytotoxicity toward the expression host result in poor soluble expression and low yields when unmodified AMPs are produced directly in prokaryotic systems. In this study, the coding sequences of three AMPs (Swine_2, P3-3R-8I, and QLX-3DV-1) were assembled by primer-complementary PCR, and nine recombinant expression vectors covering three fusion-tag systems were constructed by restriction digestion and ligation, namely pET32a-Swine_2, pET32a-P3-3R-8I, pET32a-QLX-3DV-1, pET32a-GSHHW-Swine_2, pET32a-GSHHW-P3-3R-8I, pET32a-GSHHW-QLX-3DV-1, pET3C-SUMO-Swine_2, pET3C-SUMO-P3-3R-8I, and pET3C-SUMO-QLX-3DV-1. The resulting plasmids were transformed into Escherichia coli BL21(DE3), and the nine fusion proteins were expressed under IPTG induction and auto-induction. The fusion proteins were purified by Ni-NTA affinity chromatography; the target AMPs were released by enterokinase digestion, SNAC-mediated chemical cleavage, or SUMO protease digestion, and the peptides were recovered by ultrafiltration. The antibacterial activity of the recovered peptides against E. coli and Staphylococcus aureus was systematically compared using the agar diffusion assay and the broth microdilution method. All three strategies yielded bioactive AMPs. For a given peptide backbone, the three preparations showed identical MIC values, whereas the peptides released by SUMO protease produced the largest inhibition zones. Because the three preparations of any one backbone share an identical primary sequence and an identical MIC, this difference cannot be attributed to a higher intrinsic potency of the peptide; it is treated here as a semi-quantitative observation. This study provides a complete technical workflow and a methodological reference for the engineered production of AMPs.