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 centralcomponent of human innate immunity. Its broader application is unfortunately limited due to high productioncosts, 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 proteinand an endoplasmic reticulum retention signal. The fusion protein MBP_rhLL-37 was extracted from lyophilizedimmature barley grains and enriched by ammonium sulphate precipitation. Proteolytic processing of the fusionprotein was evaluated using the epidermal serine proteases KLK5, KLK7, KLK8, and KLK14, and the antimicrobialactivity of the resulting products was assessed against Escherichia coli TOP10. Among the tested proteases, KLK14showed the most efficient cleavage of the fusion protein and generated LL-37-immunoreactive products associatedwith antimicrobial activity. In addition, the barley extract containing MBP_rhLL-37 was successfullyincorporated into a cream formulation suitable for topical delivery studies. Overall, this work demonstrates thatbarley-based molecular farming, combined with non-chromatographic purification and physiologically relevantprotease activation, provides a practical, cost-effective, and environmentally sustainable platform for the LL-37application in topical formulation.