Heterologous expression, refolding, and functional characterization of Halocin C8 from halophilic archaea in...

Halocins are antimicrobial peptides secreted by halophilic archaea, stable under hypersaline conditions and promising for food preservation and biomedical fields. In this study, bioinformatic analysis revealed that the halC8 coding region of Haloferax sp. TG-1 was 234 bp, encoding a mature Halocin C8 protein of 77 amino acids with a theoretical molecular weight of 7.513 kDa and an isoelectric point of 3.47. Phylogenetic analysis showed that distinct halocin families were phylogenetically divergent, while Halocin C8 homologs were conserved within the same genus but highly variable across genera. Subsequently, the halC8 coding region was amplified and successfully expressed in Escherichia coli BL21(DE3). Following urea-mediated solubilization of inclusion bodies and Ni-NTA affinity purification, a hypersaline buffer (4 M NaCl, 50 mM Tris-HCl, 10 mM CaCl₂) was employed to refold the recombinant Halocin C8. The refolded protein exhibited antimicrobial activity against haloarchaeal strains, particularly against Haloarcula, with a maximum inhibition zone diameter of 12.3 mm and a minimum inhibitory concentration of 0.34 mg/mL. In addition, the refolded Halocin C8 showed antioxidant activity, with a DPPH radical scavenging rate of 65.4% ± 1.3% at a concentration of 0.68 mg/mL, whereas the unfolded protein exhibited a much lower scavenging rate (27.2% ± 2.4%). This study provides a technical reference for the heterologous expression of functional halophilic proteins.