Proteolytic activation of barley-expressed human cathelicidin LL-37 by skin serine proteases

The antimicrobial peptide LL-37, the only cathelicidin present in the human body, represents a central component of human innate immunity. Its broader application is unfortunately limited due to high production costs, purification challenges, and peptide instability. This study builds on the already established, scalable, plant-based production of LL-37 in barley seeds, where it is expressed in a fusion with maltose-binding protein and an endoplasmic reticulum retention signal. The fusion protein MBP_rhLL-37 was extracted from lyophilized immature barley grains and enriched by ammonium sulphate precipitation. Proteolytic processing of the fusion protein was evaluated using the epidermal serine proteases KLK5, KLK7, KLK8, and KLK14, and the antimicrobial activity of the resulting products was assessed against Escherichia coli TOP10. Among the tested proteases, KLK14 showed the most efficient cleavage of the fusion protein and generated LL-37-immunoreactive products associated with antimicrobial activity. In addition, the barley extract containing MBP_rhLL-37 was successfully incorporated into a cream formulation suitable for topical delivery studies. Overall, this work demonstrates that barley-based molecular farming, combined with non-chromatographic purification and physiologically relevant protease activation, provides a practical, cost-effective, and environmentally sustainable platform for the LL-37 application in topical formulation.