Engineered Bacterial Biosynthesis of a Cysteine-Rich SPRR2A Fusion Protein with Dual Antibacterial and Anticancer Efficacy.

Antimicrobial peptides represent promising candidates for anticancer therapy, owing to their membrane-disrupting mechanisms. Small proline-rich protein 2A (SPRR2A), a cysteine-rich gut bactericidal peptide, exhibits potent activity against Gram-positive bacteria via membrane integrity disruption. However, its complex disulfide bond network has hindered the production of bioactive SPRR2A in prokaryotic systems. Herein, we report an efficient prokaryotic biosynthesis strategy for a bioactive SPRR2A fusion protein, Trx-SPRR2A, leveraging an Rosetta-gami (DE3) pLysS strain. The recombinant Trx-SPRR2A, obtained with a high yield (7.18 mg/L) and purity (94.5%), correctly formed five intrachain disulfide bonds and demonstrated remarkable thermal and serum stability. Functionally, Trx-SPRR2A displayed dose-dependent antibacterial activity with high selectivity toward Gram-positive bacteria. Notably, it exhibited selective cytotoxicity against MCF-7 breast cancer cells under weakly acidic conditions, a hallmark of the tumor microenvironment. Mechanistic studies revealed that the dual functionality stems from membrane disruption in both bacterial and cancer cells alongside the induction of apoptosis in MCF-7 cells. This work not only provides a robust method for producing complex disulfide-rich peptides but also highlights Trx-SPRR2A as a compelling dual-function therapeutic agent.