Recombinant production of isotope-labeled human α-defensin 5 via calmodulin fusion and insights into its expression enhancement

Human α-defensin 5 (HD5), a cysteine-rich antimicrobial peptide critical for intestinal innate immunity, has been extensively studied for its structural and functional properties. Both the reduced form (HD5red) and the oxidized form (HD5oxi) exist in vivo and exhibit distinct antimicrobial activity spectra. In this study, we developed an efficient method to overexpress recombinant HD5 in Escherichia coli (E. coli) BL21 (DE3) strain by using calmodulin (CaM), which also interacts with antimicrobial peptides, as a fusion partner. Fusion expression suppressed the degradation of HD5 and reduced its toxicity to host cells. Following purification of the fusion protein and enzymatic cleavage to release the HD5 region, we successfully obtained sufficient amounts (yielding 1.5–1.7 mg/L culture) of active recombinant HD5oxi and HD5red for various applications, including stable isotope-labeled peptides for NMR analysis. Furthermore, we investigated the protective effect of CaM fusion and the mechanism of disulfide bond formation using CD and NMR spectroscopy, structural prediction, and molecular dynamics simulations. Our results suggest that the appropriate interaction strength between CaM and the HD5 region in the fusion state is a key factor for stable production.