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, a 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) 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. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.