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.

Cadmium (Cd) is a pervasive environmental contaminant with potent cytotoxic effects in a wide range of organisms. Although autophagy and apoptosis are recognized as major cellular responses to heavy metal stress, the molecular basis of Cd-induced cell death in insects remains insufficiently understood. In this study, we used fifth-instar day-4 (5L4D) larvae of Bombyx mori and the silkworm-derived Bm-12 cell line to investigate the involvement of three core autophagy-related proteins, Bombyx mori Autophagy-related protein 5(BmATG5), Bombyx mori Autophagy-related protein 6(BmATG6), and Autophagy-related protein 8(BmATG8), in Cd-induced autophagy and apoptosis. Exposure to CdCl₂ markedly induced autophagic and apoptotic responses in both larval midgut tissue and Bm-12 cells, as demonstrated by monodansylcadaverine(MDC) staining, Lyso-Tracker Red staining, DAPI and Hoechst 33258 staining, and DNA fragmentation assays. qPCR and Western blot analyses showed significant upregulation of BmATG5, BmATG6, and BmATG8 following Cd exposure. Notably, the cleaved forms tBmATG5-N (24 kDa) and tBmATG6-C (35 and 37 kDa), as well as the lipidated form BmATG8-PE (12 kDa), accumulated substantially under Cd stress. In parallel, intracellular Ca²⁺ levels and calpain activity were significantly increased, suggesting activation of a calcium-dependent regulatory pathway. Pharmacological inhibition experiments further indicated that autophagy and apoptosis are functionally interconnected during the Cd response. Collectively, these findings demonstrate that BmATG5, BmATG6, and BmATG8, together with their processed forms, play central roles in coordinating autophagy–apoptosis crosstalk during Cd-induced cytotoxicity in Bombyx mori. This study provides new mechanistic insight into heavy metal toxicity in insects and expands our understanding of stress-induced programmed cell death during silkworm metamorphosis.