Calmodulin-tagging prevents aggregation and facilitates the proteolytic release of the recombinant human cathelicidin LL-37 by accommodating its hydrophobic regions.

The human cathelicidin LL-37 is a widely studied 37-residue antimicrobial peptide with diverse biological functions. However, its strong aggregation propensity hinders the efficient recombinant production of this peptide. Previously reported expression strategies often face limitations such as low solubility, inefficient tag removal, and/or the need for detergents, which complicate downstream sample preparation. Here, we developed a detergent-free method for producing recombinant LL-37 in Escherichia coli as a fusion protein with calmodulin (CaM), a protein capable of binding to amphiphilic peptides. CaM-tagged LL-37 exhibited markedly improved solubility compared with LL-37 fused to conventional tags, such as thioredoxin. The CaM segment was efficiently cleaved from the CaM-LL-37 fusion protein by enterokinase (EK), enabling effective purification of LL-37 in its native form. Nuclear magnetic resonance (NMR) spectroscopy demonstrated that the CaM and LL-37 regions of the fusion protein interact with each other in both the Ca2+-bound and Ca2+-free states through distinct interaction modes. The linker region containing the EK recognition site in the CaM-LL-37 fusion protein was also revealed to be solvent-exposed and flexible, which explains the enhanced cleavage efficiency. Molecular dynamics (MD) simulations further suggested that CaM forms extensive hydrophobic contacts with LL-37, which may effectively cover its aggregation-prone surfaces from the solvent. The present strategy was also applicable to the mouse ortholog of LL-37, mCRAMP, supporting its broader utility. These results imply that CaM-tagging provides a practical and mechanistically rational platform for producing aggregation-prone antimicrobial peptides in a soluble, detergent-free, and enzymatically processable form.