An improved lentiviral system for efficient expression and purification of β‐defensins in mammalian cells

β‐Defensins are a family of conserved small cationic antimicrobial peptides with different significant biological functions. The majority of mammalian β‐defensins are expressed in epididymis, and many of them are predicted to have post‐translational modifications. However, only a few of its members have been well studied due to the limitations of expressing and purifying bioactive proteins with correct post‐translational modifications efficiently. Here we developed a novel Fc tagged lentiviral system and Fc tagged prokaryotic expression systems provided new options for β‐defensins expression and purification. The novel lentiviral system contains a secretive signal peptide, an N‐terminal IgG Fc tag, a green fluorescent protein (GFP), and a puromycin selection marker to facilitate efficient expression and fast purification of β‐defensins by protein A magnetic or agarose beads. It also enables stable and large‐scale expression of β‐defensins with regular biological activities and post‐translational modification. Purified β‐defensins such as Bin1b and a novel human β‐defensin hBD129 showed antimicrobial activity, immuno‐regulatory activity, and expected post‐translational phosphorylation, which were not found in Escherichia coli ( E. coli ) in expressed form. Furthermore, we successfully applied the novel system to identify mBin1b interacting proteins, explaining Bin1b in a better way. These results suggest that the novel lentiviral system is a powerful approach to produce correct post‐translational processed β‐defensins with bioactivities and is useful to identify their interacting proteins. This study has laid the foundation for future studies to characterize function and mechanism of novel β‐defensins.